Rationally designed polysaccharide lyases derived from chondroitinase B

ABSTRACT

The invention relates to rationally designed polysaccharide lyases and uses thereof. In particular, the invention relates to modified chondroitinase B. The modified chondroitinase B enzymes of the invention are useful for a variety of purposes, including cleaving and sequencing polysaccharides such as glycosaminoglycans (GAGs) as well as removing polysaccharides from a solution. The invention also includes methods of inhibiting anticoagulant activity, inhibiting angiogenesis, treating cancer, and inhibiting maternal malarial infection.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 from U.S.provisional application Ser. No. 60/385,509 filed Jun. 3, 2002, theentire contents of which is incorporated by reference.

GOVERNMENT SUPPORT

Aspects of the invention may have been made using funding from NationalInstitutes of Health Grant number Grant GM 57073. Accordingly, theGovernment may have rights in the invention.

FIELD OF THE INVENTION

The invention relates to rationally designed polysaccharide lyases anduses thereof. In particular, the invention relates to modifiedchondroitinase B. The modified chondroitinase B enzymes of the inventionare useful for a variety of purposes, including cleaving and sequencingpolysaccharides such as glycosaminoglycans (GAGs) as well as removingpolysaccharides from a solution and therapeutic methods such asinhibiting anticoagulant activity, inhibiting angiogenesis, treatingcancer, and inhibiting maternal malarial infection.

BACKGROUND OF THE INVENTION

Glycosaminoglycans (GAGs) are linear, acidic polysaccharides that existubiquitously in nature as residents of the extracellular matrix and atthe cell surface of many different organisms of divergent phylogeny(Habuchi, O. (2000) Biochim Biophys Acta 1474, 115-27; Sasisekharan, R.,Bulmer, M., Moremen, K. W., Cooney, C. L., and Langer, R. (1993) ProcNatl Acad Sci USA 90, 3660-4). In addition to a structural role, GAGsact as critical modulators of a number of biochemical signaling events(Tumova, S., Woods, A., and Couchman, J. R. (2000) Int J Biochem CellBiol 32, 269-88) requisite for cell growth and differentiation, celladhesion and migration, and tissue morphogenesis.

Dermatan sulfate (DS) and chondroitin sulfate (CS) are relatedglycosaminoglycans (GAGs) that are composed of a disaccharide repeatunit of uronic acid (1→3)-linked to N-acetyl-D-galactosamine (GalNAc).These disaccharide repeats are (1→4)-linked to each other to formpolymers of chondroitin sulfate or dermatan sulfate. Epimerization atthe C5 position of the uronic acid moiety during the biosynthesis ofdermatan sulfate leads to a mixture of L-iduronic and D-glucuronic acidepimers (Ernst, S., Langer, R., Cooney, C. L., and Sasisekharan, R.(1995) Crit. Rev. Biochem. Mol. Biol. 30, 387-444). In addition to C5epimerization, C4 sulfation of GalNAc is another hallmark modificationof the DS backbone. Rare sulfation at the 2-0 and 3-0 positions of theuronic acid moiety has also been reported (Sugahara, K., Tanaka, Y.,Yamada, S., Seno, N., Kitagawa, H., Haslam, S. M., Morris, H. R., andDell, A. (1996) J. Biol. Chem. 271, 26745-54; Nadanaka, S., andSugahara, K. (1997) Glycobiology 7, 253-63). CS/DS polysaccharides havebeen implicated in a variety of biological phenomena ranging fromanticoagulation to osteoarthritis (Mascellani, G., Liverani, L.,Bianchini, P., Parma, B., Torri, G., Bisio, A., Guerrini, M., and Casu,B. (1993) Biochem. J. 296, 639-48; Achur, R. N., Valiyaveettil, M.,Alkhalil, A., Ockenhouse, C. F., and Gowda, D. C. (2000) J. Biol. Chem.275, 40344-56; and Plaas, A. H., West, L. A., Wong-Palms, S., andNelson, F. R. (1998) J. Biol. Chem. 273, 12642-9). In fact, specificsequences of highly sulfated dermatan sulfate from a variety ofinvertebrate and mammalian sources are being pursued as pharmaceuticallyviable treatments for specific blood coagulation disorders (Monagle, P.et al. (1998) J. Biol. Chem. 273, 33566-71; Gandra, M. et al. (2000)Glycobiology 10, 1333-40; and Vicente, C. P. et al. (2001) Thromb.Haemost. 86, 1215-20). Changes in the dermatan sulfate side chain of thesmall proteoglycan, decorin, have been observed in human colon cancer(Daidouji, K. et al. (2002) Dig. Dis. Sci. 47, 331-7). And modificationof existing GAG sequences by chondroitinase B and chondroitinase AC mayinhibit angiogenesis and tumor metastasis (Denholm, E. M. et al. (2001)Eur. J. Pharmacol. 416, 213-21). Overall, the role of GAGs as specificmediators of tumorigenesis and other biological events is an emergingfield that offers great potential for the development of noveltherapeutics (Shriver, Z. et al. (2002) Trends. Cardiovasc. Med. 12,71-7; and Liu, D. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 568-73).

Flavobacterium heparinum is a common source for GAG-degrading lyases,producing both the extensively characterized heparin-degradingheparinases (Sasisekharan, R., Venkataraman, G., Godavarti, R., Ernst,S., Cooney, C. L., and Langer, R. (1996). J. Biol. Chem. 271, 3124-31;Shriver, Z., Hu, Y., Pojasek, K., and Sasisekharan, R. (1998) J. Biol.Chem. 273, 22904-12; Pojasek, K., Shriver, Z., Hu, Y., and Sasisekharan,R. (2000) Biochemistry 39, 4012-9; and Gu, K., Linhardt, R. J.,Laliberte, M., and Zimmermann, J. (1995) Biochem. J. 312, 569-77), aswell as the CS/DS-degrading chondroitinases (Gu, K. et al. (1995)Biochem. J. 312, 569-77). Chondroitinase B is the only member of thechondroitinase family that degrades DS as its sole substrate (Jandik, K.A., Gu, K., and Linhardt, R. J. (1994) Glycobiology 4, 289-96 andPojasek, K., Shriver, Z., Kiley, P., Venkataraman, G., and Sasisekharan,R. (2001) Biochem. Biophys. Res. Commun. 286, 343-51).

SUMMARY OF THE INVENTION

The present invention relates, in part, to modified polysaccharidelyases. In particular, the invention relates to modified chondroitinaseB. The characterization of the chondroitinase B active site,specifically the individual residues involved in substrate binding andcatalysis allows for the rational design of modified chondroitinase Benzymes described herein. Additionally, the modified enzymes may be usedfor a variety of purposes due to the ability of the enzymes to uniquelycleave polysaccharides such as the glycosaminoglycans chondroitinsulfate and dermatan sulfate, or compete with native enzyme forsubstrate.

The invention, therefore, in some aspects is a modified chondroitinase Bhaving an amino acid sequence of the mature peptide of SEQ ID NO: 2 orconservative substitutions thereof, wherein at least one residue at aposition selected from the group consisting of 116, 184, 213, 219, 245,250, 271, 272, 296, 298, 318, 333, 363 and 364 of SEQ ID NO: 2 has beensubstituted or deleted. In other embodiments the modified chondroitinaseB has the amino acid sequence of the mature peptide of SEQ ID NO: 2wherein at least one amino acid residue has been substituted and whereinthe substituted amino acid is at a position selected from the groupconsisting of 272, 333, and 364 of SEQ ID NO: 2. In still otherembodiments the modified chondroitinase B has the amino acid sequence ofthe mature peptide of SEQ ID NO: 2 wherein at least one amino acidresidue has been substituted and wherein the substituted amino acid isat a position selected from the group consisting of 272, 333, 363 and364 of SEQ ID NO: 2. In further embodiments, the modified chondroitinaseB has the amino acid sequence of the mature peptide of SEQ ID NO: 2wherein at least one residue has been substituted and wherein thesubstituted amino acid is at position 364 of SEQ ID NO: 2. In anotheraspect, modified chondroitinase B enzymes contain at least onesubstitution but maintain one or more of the residues with binding orcatalytic activity recited herein. In one embodiment, the residue isresidue at position 116, 184, 213, 219, 245, 250, 271, 272, 296, 298,318, 333, 363 or 364 of SEQ ID NO: 2. In another embodiment the residueis at position 250 of SEQ ID NO: 2.

The modified chondroitinase B enzymes may also be described as having amodified product profile due to the interaction of the enzyme withsubstrate. The invention in some aspects is a modified chondroitinase Bhaving a modified product profile, wherein the modified product profileof the modified chondroitinase B is at least 10% different than a nativeproduct profile of a native chondroitinase B. In other embodiments themodified product profile of the modified chondroitinase B is at least50% different than a native product profile of a native chondroitinaseB. In still other embodiments the modified product profile is at least20% different than a native product profile of a native chondroitinaseB.

In other aspects a modified chondroitinase B having a k_(cat) or K_(M)value for a substrate that is at least 10% different than a nativechondroitinase B k_(cat) or K_(M) value is provided. In otherembodiments the k_(cat) or K_(M) value is at least 20% different than anative chondroitinase B k_(cat) or K_(M) value. In still otherembodiments the k_(cat) or K_(M) value is at least 50% different than anative chondroitinase B k_(cat) or K_(M) value.

The invention in some aspects also provides an enzyme, characterized byan active site organized in a three dimensional space along an axiscomposed of 4 regions identified as −2, −1, +1, and +2 and including atleast the following amino acid residues positioned along the axis at thedefined points 4 basic amino acids and 1 polar amino acid in −2 region,2 basic amino acids and 1 acidic amino acid in −1 region and +1 region,and 2 basic amino acids in +2 region, wherein the enzyme does not havethe primary sequence of native chondroitinase B. In some embodiments ofthe invention the enzyme includes at least the following amino acidresidues positioned along the axis at the defined points 4 Arg, and 1Phe in −2 region, 1 Asn, 1 Glu, and 1 Arg in −1 region, 1 Lys, 1 Glu,and 1 His in +1 region, and 1 His and 1 Arg in +2 region. In still otherembodiments the enzyme comprises 1 Arg and 1 Trp in −1 region.

In some embodiments of the aforementioned enzymes, the substituted aminoacid is a conservative amino acid substitution. In other embodiments,the enzyme is a substantially purified recombinant form. In someembodiments the substrate for the enzyme is a polysaccharide. In stillother embodiments the substrate is a long polysaccharide. In still otherembodiments the polysaccharide is a decasaccharide. In yet otherembodiments the polysaccharide is an octa-, hexa- or tetrasaccharide. Inyet another embodiment the substrate for the enzymes is aglycosaminoglycan.

The modified chondroitinase B and preparations may be utilized forvarious purposes. In some aspects a method of specifically cleavingchondroitin sulfate, comprising contacting chondroitin sulfate with themodified chondroitinase B is provided. In other embodiments the methodis a method of specifically cleaving dermatan sulfate. In otherembodiments a method of removing chondroitin sulfate from a chondroitinsulfate containing fluid is provided. In still other embodiments themethod is a method of removing dermatan sulfate from a dermatan sulfatecontaining fluid. The method is, in some embodiments, a method forsequencing chondroitin sulfate oligosaccharides. In other embodimentsthe method is a method for sequencing dermatan sulfate oligosaccharides.The invention also provides in some aspects an immobilized modifiedchondroitinase B comprising a modified chondroitinase and a solidsupport membrane, wherein the modified chondroitinase B is immobilizedon the solid support membrane.

In some aspects a method of analyzing a sample of polysaccharides,comprising contacting the sample with the modified chondroitinase B isprovided. Another aspect is a method of identifying the presence of aparticular polysaccharide in a sample. In still other aspects a methodof determining the purity of sample of polysaccharides is provided. Inyet other aspects a method for determining the composition of a sampleof polysaccharides is provided.

In some aspects the invention relates to a method for purifying orisolating a recombinant enzyme. In some embodiments the recombinantenzyme is a polysaccharide degrading enzyme. In still another embodimentthe recombinant enzyme is a chondroitinase. The method may involve theinduction of a culture of cells containing a recombinant chondroitinasewith an inducing agent for greater than four hours, followed byisolation of the recombinant chondroitinase from the cells to produce apurified chondroitinase. The method may also involve lysing a cellculture containing a recombinant chondroitinase having a terminalHistidine tag, and passing the recombinant chondroitinase over a chargedNi 2+ column to isolate the recombinant chondroitinase. According to yetother embodiments the inducing agent isisopropyl-B-D-thiogalactopyranoside (IPTG).

In some embodiments the cells are incubated with the inducing agent at atemperature of between 20° and 26° C. In other embodiments the cells areincubated with the inducing agent for at least 8 hours. In yet otherembodiments the chondroitinase is chondroitinase AC or B. In yet otherembodiments, the chondroitinase is a modified chondroitinase B.

The modified chondroitinase B or glycosaminoglycan fragment producedwith the modified chondroitinase B is also useful for therapeuticpurposes. The method in some embodiments is directed to modulating acondition with the modified chondroitinase B or glycosaminoglycanfragment. The invention in some embodiments is a method for inhibitingangiogenesis, by administering to a subject an effective amount ofchondroitinase B for inhibiting angiogenesis. In other embodiments thechondroitinase B is administered directly to a tumor. A method forinhibiting maternal malarial infection, by administering to a subject inneed thereof an effective amount for maternal malarial infection of themodified chondroitinase B is also provided. In some embodiments a methodfor inhibiting anticoagulant activity of dermatan sulfates, comprisingadministering to a subject in need thereof an effective amount forinhibiting anticoagulant activity of dermatan sulfates of the modifiedchondroitinase B is also provided. In still other embodiments a methodfor treating osteoarthritis is provided. In other embodiments a methodfor treating cancer, by administering to a subject in need thereof aneffective amount for treating cancer of the modified chondroitinase B isprovided. In still other embodiments the cancer is metastatic cancer. Inyet other embodiments methods for modulating mitogenic activity (e.g.FGF-7 mitogenic activity), enhancing hepatocyte growth factor/scatterfactor activity and mediating cell signaling are provided.

In some embodiments a pharmaceutical preparation is provided comprisinga sterile formulation of chondroitinase B and a pharmaceuticallyacceptable carrier. In other embodiments a pharmaceutical preparation isprovided comprising a glycosmaminoglycan fragment. In other embodimentsthe pharmaceutical preparation comprises a combination of differentglycosaminoglycan fragments. Glycosaminoglycan fragments can be producedby the action of a modified chondroitinase B alone or in combinationwith other enzymes. In other embodiments the chondroitinase B isadministered in a biodegradable, biocompatible polymeric deliverydevice. In still other embodiments the chondroitinase B orglycosaminoglycan fragment is administered in a pharmaceuticallyacceptable vehicle for injection.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the docking of the dermatan sulfate substrate in theactive site of chondroitinase B. (A) Stereoview of conolly surfacerendering of the active site of chondroitinase B with the dockeddermatan sulfate tetrasaccharide (green) and disaccharide product(orange) whose orientation is replicated from the co-crystal structure.Although the direction of both the disaccharide product and thetetrasaccharide is the same from non-reducing end (close to C terminusabove active site) to reducing end (close to N-terminus below the activesite), the tetrasaccharide is positioned to completely occupy the activesite. (B) Stick representation of the dermatan sulfate tetrasaccharidein the active site of chondroitinase B, colored according to the atoms(C: green, N: blue, O: red and S: yellow) (left) and the two dimensionalschematic distribution of the active site residues (right). The sidechains of the residues (single letter code and number) of the proteininteracting with the tetrasaccharide are shown. Basic residues (Lys,Arg, Asn, His) are colored blue, acidic residues (Glu) are colored red,and bulky aromatic residues (Phe, Trp) are colored purple. The subsitenomenclature is used to define the orientation of the tetrasaccharidefrom −2 (nonreducing end) to +2 (reducing end) in the active site.Cleavage occurs between the −1 and +1 site.

FIG. 2 details the apparent internal symmetry in the active site ofchondroitinase B. The grasp rendered view of the active site is shown onthe left with the basic residues (H, K, R) colored in blue, acidicresidues (D, E) colored in red, and bulky hydrophobic residue (F, Y, W)colored in pink. On the right is a two dimensional schematic of theresidues with their sequence numbers encircled using the same colorcoding scheme as on the left. Also shown on the right is an arrowcolored (gray) indicating the assumed direction of the dermatan sulfatein this study (point of arrow indicates the reducing end). There is anapproximate two-fold symmetry in the distribution of the acidic, basicand, hydrophobic residues about an axis perpendicular to the helix ofthe dermatan sulfate oligosaccharide.

FIG. 3 summarizes the capillary electrophoretic analysis of the dermatansulfate reaction products for the catalytic mutations. (A) Recombinantchondroitinase B (20 μg), (B) H272A, (C) E333A, and (D) K250A wereincubated with the 1 mg/ml dermatan sulfate for 12 hr at 30° C.Capillary electrophoretic analysis was performed using an extendedpath-length cell and a voltage of 30 kV applied using reverse polarity.Saccharides were injected into the capillary using hydrodynamic pressureand were detected using an ultraviolet detector set at 232 nm. Therunning buffer consisted of 50 mM Tris, 10 μM dextran sulfate that hadbeen brought to a pH of 2.5 using phosphoric acid. The disulfateddisaccharides, ΔUA-GalNAc2S4S and ΔUA-GalNAc4S,6S, are indicated by “*”and “**”, respectively. (inset) Electropherogram of the ΔUA-GalNAc4Sdisaccharide standard.

FIG. 4 summarizes the capillary electrophoretic analysis of the reactionproducts for the substrate binding mutations. (A) R363A and (B) R364Awere incubated with 1 mg/ml dermatan sulfate for 12 hr at 30° C. andanalyzed using capillary electrophoresis. The length and sulfatecomposition of the additional peaks in the R364A digest (B) weredetermined using MALDI-MS. Peak 1 is an octasaccharide (1922.4 Da) with5 sulfates. Peak 2 is a hexasaccharide (1539.7 Da) with 5 sulfates. AndPeak 3 is a tetrasaccharide (999.2 Da) with 3 sulfates. The disulfateddisaccharides, ΔUA-GalNAc2S,4S and ΔUA-GalNAc4S,6S, are indicated by “*”and “**”, respectively.

FIG. 5 provides the CD spectra of chondroitinase B and the K250A mutant.The recombinant chondroitinase B (●) and the K250A mutant (∘) wereconcentrated and buffer exchanged into 50 mM sodium phosphate buffer, pH7.0. Proteins were analyzed in a quartz cell with 1 mm path length at25° C. CD Spectra were recorded between 200 and 270 nm with an averageof 5 scans; the bandwidth was set 1.0 nm; and the scan rate was 3nm/min. The CD band intensities are expressed as molar ellipticities,θ_(M), in deg·cm²·dmol⁻¹.

FIG. 6 illustrates the generation and purification of defined DSoligosaccharides. DS was partially digested with the chondroitinase Bmutant, R364A, and the products were separated on a Bio-gel P6 column.(A) Six distinct peaks with absorbance at 232 nm were pooled,lyophilized, and further separated using HPLC. Each peak was analyzedusing capillary electrophoresis and MALDI-MS to assess their purity andto assign their identity. (B) A representative electropherogram of theDS oligosaccharide from peak 2 confirms its purity. (C) The majoroligosaccharide from peak 2 was complexed with the basic peptide,(arg-gly)₁₅, and analyzed using MALDI-MS. Subtracting the mass of thepeptide (3218.9 Da) from the mass of the oligosaccharide:peptide complex(5515.9 Da) yielded an oligosaccharide with a mass of 2297.0 Da,identifying peak 2 as a decasaccharide with 5 sulfates. Peak 1 wasidentified as a dodecasaccharide with 6 sulfates, peak 3 was anoctasaccharide with 4 sulfates, peak 4 was a hexasaccharide with 3sulfates, peak 5 was a tetrasaccharide with 2 sulfates, and peak 6 was amono-sulfated disaccharide.

FIG. 7 provides the structure of relevant DS oligosaccharides. (A) Afive-sulfated decasaccharide derived from the partial enzymatic digestof DS. The decasaccharide is characterized by sulfates at the 4-Oposition of each GalNAc, IdoA epimers of the uronic acids, and a Δ^(4.5)unsaturated double bond at the non-reducing end. (B) A three-sulfatedhexasaccharide derived from the partial enzymatic digest of DS. (C) Thesame hexasaccharide as in (B) with a semicarbazide mass tag attached toits reducing end. The presence of the semicarbazide label enabledtracking of the reducing end disaccharide during the enzymaticdegradation by capillary electrophoresis and MALDI-MS. Thedecasaccharide in (A) was also labeled in a similar fashion. (D) Aschematic representation of the semicarbazide labeled hexasaccharide in(C). The triangle represents the non-reducing end 4-sulfateddisaccharide with the Δ^(4.5) double bond. Each circle is a 4-sulfateddisaccharide and the star represents the semicarbazide label on thereducing end of the oligosaccharide. The arrows indicate potentialcleavable bonds at site I and site II. (E) A schematic representation ofa semicarbazide labeled decasaccharide. The shapes are the same asdescribed for the hexasaccharide in (D). The decasaccharide has fourcleavable bonds; two terminal, exolytic bonds (site I and IV) and twointernal, endolytic bonds (site II and III).

FIG. 8 provides results of chondroitinase B degradation of Deca.Chondroitinase B was incubated with the five-sulfated decasaccharide fordefined period of times, and the enzymatic products were analyzed by CE.The resulting peak areas in the electropherogram were converted to molarconcentrations and plotted versus time. (A) During the 120 min.digestion of Deca (□), there was an initial appearance of Tetra (▬) andHexa (▴) with very little Octa (x) and Di (♦) products indicating thatchondroitinase B is an endolytic enzyme. (B) This observation wasconfirmed by examining the products of the enzymatic reaction during thefirst 60 s. Later in the reaction time course as Deca was depleted (A),the concentration of Hexa decreased with a concomitant increase in Diand Tetra, implying that chondroitinase B prefers longer substrates(Deca) to shorter ones (Hexa).

FIG. 9 provides results from the digestion of Hexa-sc. A hexasaccharidelabeled at the reducing end was digested with chondroitinase B (A) andthe R364A mutant (B) and analyzed using capillary electrophoresis. (A)The initial reaction products resulting from the digestion of theHexa-sc (H-sc) substrate by chondroitinase B are Tetra (T), Tetra-sc(T-sc), Di and Di-sc. (B) There was a noticeable increase in therelative concentration of T-sc and Di produced when H-sc was degraded byR364A, suggesting that this mutant has an altered mode of action whencompared to chondroitinase B. (* denotes the remaining unlabeled Hexaimpurity from the semicarbazide labeling)

FIG. 10 provides the results of the digestion of Deca-sc. Adecasaccharide labeled at the reducing end with semicarbazide wasdigested with chondroitinase B (A) and the R364A mutant (B) and analyzedby capillary electrophoresis. (A) The major products of the digestion ofDeca-sc (D-sc) were Hexa-sc (H-sc), Hexa (H), Tetra-sc (T-sc), and Tetra(T). The higher relative amounts of T and H-sc indicate thatchondroitinase B acts in a non-random fashion, preferring to cleave theinternal bond proximal to the reducing end to the internal bond nearestthe non-reducing end. (B) Digestion of D-sc with the R364A mutantproduces the same products as in the chondroitinase B digestion.However, the relative amount of each product is different implying thatthe R364A mutant has lost the non-random aspect of the mode of action,thus cleaving both internal bonds with near equal efficiency. (* denotesthe remaining unlabeled Deca impurity from the semicarbazide labeling)

DETAILED DESCRIPTION

Dermatan sulfate (DS) is a member of the glycosaminoglycan (GAG) familyof complex polysaccharides that also includes chondroitin sulfate (CS),heparin/heparan sulfate (HSGAG), keratan sulfate, and hyaluronic acid.Chondroitin sulfate and dermatan sulfate glycosaminoglycanpolysaccharides, have been implicated in biological processes rangingfrom osteoarthritis to anticoagulation. Dermatan sulfate is emerging asan important regulator of cellular signaling processes. An over-sulfatedhexasaccharide found in DS that binds heparin cofactor II and promotes a1000-fold increase in anticoagulation is the most characterizedbiological paradigm for DS (Maimone, M. M., and Tollefsen, D. M. (1991)J Biol Chem 266, 14830; Mascellani, G., Liverani, L., Bianchini, P.,Parma, B., Torri, G., Bisio, A., Guerrini, M., and Casu, B. (1993)Biochem J 296, 639-48). Several recent studies have implicated DS inpromoting FGF-7 mitogenic activity (Trowbridge, J. M., Rudisill, J. A.,Ron, D., and Gallo, R. L. (2002) J Biol Chem 277, 42815-20) andenhancing the activity of hepatocyte growth factor/scatter factor (Lyon,M., Deakin, J. A., Rahmoune, H., Fernig, D. G., Nakamura, T., andGallagher, J. T. (1998) J Biol Chem 273, 271-8; Lyon, M., Deakin, J. A.,and Gallagher, J. T. (2002) J Biol Chem 277, 1040-6), suggesting animportant role for DS in mediating cell signaling. One of the majorhurdles in studying the biochemistry of DS as well as the other GAGs hasbeen dealing with their overall structural heterogeneity and negativecharge (Ernst, S., Langer, R., Cooney, C. L., and Sasisekharan, R.(1995) Crit Rev Biochem Mol Biol 30, 387-444).

Found as a proteoglycan linked to a variety of core proteins on the cellsurface or in the extracellular matrix, DS chains are composed adisaccharide repeat of a uronic acid α/β(1→3)-linked to aN-acetyl-D-galactosamine (GalNAc). Each disaccharide unit is, in turn,β(1→4)-linked to an adjacent disaccharide forming the DS chain(Trowbridge, J. M., and Gallo, R. L. (2002) Glycobiology 12, 117R-25R).The hallmark modification of DS is sulfation at the 4-O position of theGalNAc with sulfation also occurring at the 2-O position of the uronicacid and 6-O position of the GalNAc and rare sulfation at the 3-Oposition of the uronic acid (Ernst, S., Langer, R., Cooney, C. L., andSasisekharan, R. (1995) Crit Rev Biochem Mol Biol 30, 387-444; Sugahara,K., Tanaka, Y., Yamada, S., Seno, N., Kitagawa, H., Haslam, S. M.,Morris, H. R., and Dell, A. (1996) J Biol Chem 271, 26745-54). Inaddition, the uronic acid can be epimerized at the C5 position fromglucuronic acid (GlcA) to iduronic acid (IdoA) leading to furtherstructural heterogeneity (Ernst, S., Langer, R., Cooney, C. L., andSasisekharan, R. (1995) Crit Rev Biochem Mol Biol 30, 387-444).

Polysaccharide lyases have important utility not only for elucidatingthe structure and function of these glycosaminoglycans but also fortherapeutic purposes due to their cleavage of these substrates.Chondroitinase B from Flavobacterium heparinum is the only known lyasethat cleaves dermatan sulfate as its sole substrate (Ernst, S., Langer,R., Cooney, C. L., and Sasisekharan, R. (1995) Crit Rev Biochem Mol Biol30, 387-444). The sequence of chondroitinase B is well known in the art.For instance, GenBank Accession number U27584 provides the nucleic acidand amino acid sequence of chondroitinase B from Flavobacteriumheparinum. SEQ ID NO: 1 is the nucleic acid of chondroitinase B, whileSEQ ID NO: 2 provides the amino acid sequence. The GenBank recordfurther provides the sequences of the signal and mature peptides. The“mature peptide” is the sequence of chondroitinase B sans the signalpeptide sequence. The nucleic acid and amino acid sequences ofchondroitinase B from Flavobacterium heparinum are also provided is U.S.Pat. Nos. 6,054,569 and 6,093,563, issued Apr. 25, 2000 and Jul. 25,2000, respectively. Additional information from crystal structures ofchondroitinase B are also provided in GenBank (e.g. GenBank Accessionnumbers 1DBOA and 1DBGA).

GAG-degrading lyases, such as chondroitinase B, from F. heparinum arethought to cleave their DS substrates through a concerted β-eliminationmechanism originally proposed by Gassinan and Gerlt (Gerlt, J. A., andGassman, P. G. (1993) Biochemistry 32, 11943-52). The first step in theproposed reaction is the abstraction of the C5 proton on the GalNAcmoiety by a basic amino acid forming an enolate intermediate. The enzymestabilizes this carbanion intermediate usually via a positively charged,hydrophilic amino acid (Gerlt, J. A., and Gassman, P. G. (1993)Biochemistry 32, 11943-52 and Gacesa, P. (1992) Int. J. Biochem. 24,545-52). The final step of reaction mechanism involves protonation ofthe anomeric oxygen by an acidic residue with concomitant β-eliminationof the uronic acid resulting an unsaturated Δ^(4.5) bond (Gerlt, J. A.,and Gassman, P. G. (1993) Biochemistry 32, 11943-52 and Gacesa, P.(1992) Int. J. Biochem. 24, 545-52).

The roles of specific active site amino acids in the catalytic functionof chondroitinase B were assessed by docking a dermatan sulfatetetrasaccharide into the proposed active site of the enzyme. Ourconformational analysis also revealed a unique, symmetrical arrangementof active site amino acids that may impinge on the catalytic mechanismof action of chondroitinase B. The catalytic residues, Lys250, Arg271,His272, and Glu333 along with the substrate binding residues, Arg363 andArg364, were mutated using site-directed mutagenesis, and the kineticsand product profile of each mutant were compared to recombinantchondroitinase B. Mutating Lys250 to alanine resulted in inactivation ofthe enzyme, potentially attributable to the residue's role instabilizing the carbanion intermediate formed during enzymaticcatalysis. The His272 and Glu333 mutants showed diminished enzymaticactivity that could be indicative of a possible role for one or bothresidues in the abstraction of the C5 proton from the galactosamine. Inaddition, the Arg364 mutant had an altered product profile afterexhaustive digestion of dermatan sulfate suggesting a role for thisresidue in defining the substrate specificity of chondroitinase B. TheArg364 mutant exhibited altered the enzyme's kinetic activity likelythrough changes in substrate binding. This demonstrates an altered modeof action pattern confirming this residue's role in substrateprocessing.

Several discoveries described herein therefore contribute to themolecular understanding of chondroitinase B depolymerization of CS/DSoligosaccharides. Based on our molecular characterization ofchondroitinase B, both H272A and E333A showed altered kinetics whencompared with the recombinant chondroitinase B. Both of these mutationslead to a slight reduction in K_(m) while drastically reducing k_(cat).In addition to kinetic analysis, each of the mutant enzymes and therecombinant chondroitinase B were allowed to exhaustively digestdermatan sulfate to determine changes in product profile. A comparisonbetween the ratio of the ΔUA-GalNAc4S peak to the total peak area of themutant digests and the recombinant enzyme showed that H272A and E333Ademonstrated full enzymatic activity suggesting that, while His272 andGlu333 are important in the active site chemistry, chondroitinase B canstill function without them. The His272 and Glu333 mutants' diminishedenzymatic activity could be indicative of a possible role for one orboth residues in the abstraction of the C5 proton from thegalactosamine. Changing Lys250, however, to alanine ablated the activityof chondroitinase B suggesting that Lys250 is important for thecatalytic activity of chondroitinase B, likely attributable to theresidue's role in stabilizing the carbanion intermediate formed duringenzymatic catalysis. Along with the active site residues discussedabove, Arg271 was mutated to alanine. The R271A mutant was expressed atcomparable levels to the recombinant chondroitinase B, but wascompletely insoluble. Taken together, these results suggest that Lys250,His272, Glu333, and possibly Arg271 are involved in the catalyticdegradation of dermatan sulfate by chondroitinase B.

In addition to catalytic residues, two basic residues proximal tosubsites −1 and 2, Arg363 and Arg364, were selected for mutagenesis. TheR363A mutant had a two-fold increase in k_(cat)/K_(m) which suggeststhat removal of Arg363 allows for a slight increase in catalyticefficiency in chondroitinase B. In contrast, mutating Arg364 to alanineled to a loss of activity in the real-time kinetic assay and an alteredproduct profile after exhaustive digestion of dermatan sulfate. Assuggested by our analyses, Arg364 is important for the proper substratebinding and digestion of dermatan sulfate by chondroitinase B. Fromcompositional analysis it also appears that Arg364 is involved inchondroitinase B's ability to recognize and cleave regions containingΔUAGalNAc4S,6S in dermatan sulfate.

One of ordinary skill in the art is enabled, in light of the presentdisclosure, to produce modified chondroitinase B by standard technology,including recombinant technology, direct synthesis, mutagenesis, etc.For instance, one may produce the modified chondroitinase B having anamino acid sequence of the mature peptide of SEQ ID NO: 2 orconservative substitutions thereof, wherein at least one residue at aposition selected from the group consisting of 116, 184, 213, 219, 245,250, 271, 272, 296, 298, 318, 333, 363 and 364 of SEQ ID NO: 2 has beensubstituted or deleted. One of skill in the art may also substituteappropriate codons to produce the desired amino acid substitutions inSEQ ID NO:2 by standard site-directed mutagenesis techniques. It ispossible to use any sequence which differs from the nucleic acidequivalents of SEQ ID NO:2 only due to the degeneracy of the geneticcode as the starting point for site directed mutagenesis. The mutatednucleic acid sequence may then be ligated into an appropriate expressionvector and expressed in a host such as F. heparinum or E. coli. Theresultant modified chondroitinase B may then be purified by techniquesknown by those of ordinary skill in the art, including those disclosedbelow.

In some embodiments the modified chondroitinase B is in substantiallypure form. As used herein, the term “substantially pure” means that theproteins are essentially free of other substances to an extent practicaland appropriate for their intended use. In particular, the proteins aresufficiently pure and are sufficiently free from other biologicalconstituents of their hosts cells so as to be useful in, for example,protein sequencing, or producing pharmaceutical preparations.Polypeptides can be isolated from biological samples, and can also beexpressed recombinantly in a variety of prokaryotic and eukaryoticexpression systems by constructing an expression vector appropriate tothe expression system, introducing the expression vector into theexpression system, and isolating the recombinantly expressed protein.Polypeptides can also be synthesized chemically using well-establishedmethods of peptide synthesis. In some embodiments, chondroitinase B in asubstantially purified recombinant form is a preparation of modifiedchondroitinase B which has been recombinantly synthesized and which isgreater then 90% free of contaminants. Preferably, the material isgreater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even greaterthen 99% free of contaminants. The degree of purity may be assessed bymeans known in the art.

As used herein with respect to polypeptides, “isolated” means separatedfrom its native environment and present in sufficient quantity to permitits identification or use. Isolated, when referring to a protein orpolypeptide, means, for example: (i) selectively produced by expressioncloning or (ii) purified as by chromatography or electrophoresis.Isolated proteins or polypeptides may be, but need not be, substantiallypure. Because an isolated polypeptide may be admixed with apharmaceutically acceptable carrier in a pharmaceutical preparation, thepolypeptide may comprise only a small percentage by weight of thepreparation. The polypeptide is nonetheless isolated in that it has beenseparated from the substances with which it may be associated in livingsystems, i.e., isolated from other proteins.

A “modified chondroitinase B polypeptide” is a polypeptide whichcontains one or more modifications to the primary amino acid sequence ofa chondroitinase B polypeptide. Modifications which create a modifiedchondroitinase B polypeptide may be made recombinantly to the nucleicacid which encodes the modified chondroitinase B polypeptide, and caninclude deletions, point mutations, truncations, amino acidsubstitutions and addition of amino acids or non-amino acid moieties to(as described herein): 1) alter enzymatic activity; 2) provide a novelactivity or property to a modified chondroitinase B polypeptide, such asaddition of a detectable moiety; or 3) to provide equivalent, greater orlesser interaction with other molecules (e.g., chondroitin sulfate anddermatan sulfate). Alternatively, modifications can be made directly tothe polypeptide, such as by cleavage, and the like. Modifications alsoembrace fusion proteins comprising all or part of the modifiedchondroitinase B amino acid sequence.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Modified polypeptides are then expressed and tested forone or more activities to determine which mutation provides a modifiedpolypeptide with the desired properties.

Methods for making amino acid substitutions, additions or deletions arewell known in the art. The terms “conservative substitution”,“non-conservative substitutions”, “non-polar amino acids”, “polar aminoacids”, and “acidic amino acids” are all used consistently with theprior art terminology. Each of these terms is well-known in the art andhas been extensively described in numerous publications, includingstandard biochemistry text books, such as “Biochemistry” by GeoffreyZubay, Addison-Wesley Publishing Co., 1986 edition, which describesconservative and non-conservative substitutions, and properties of aminoacids which lead to their definition as polar, non-polar or acidic.

One type of amino acid substitution is referred to as a “conservativesubstitution.” As used herein, a “conservative amino acid substitution”or “conservative substitution” refers to an amino acid substitution inwhich the substituted amino acid residue is of similar charge as thereplaced residue and is of similar or smaller size than the replacedresidue. Conservative substitutions of amino acids include substitutionsmade amongst amino acids within the following groups: (a) the smallnon-polar amino acids, A, M, I, L, and V; (b) the small polar aminoacids, G, S, T and C; (c) the amido amino acids, Q and N; (d) thearomatic amino acids, F, Y and W; (e) the basic amino acids, K, R and H;and (f) the acidic amino acids, E and D. Substitutions which are chargeneutral and which replace a residue with a smaller residue may also beconsidered “conservative substitutions” even if the residues are indifferent groups (e.g., replacement of phenylalanine with the smallerisoleucine). The term “conservative amino acid substitution” also refersto the use of amino acid analogs or variants.

Additionally, some of the amino acid substitutions are non-conservativesubstitutions. Non-conservative substitutions, such as between, ratherthan within, the above groups (or two other amino acid groups not shownabove), which will differ more significantly in their effect onmaintaining (a) the structure of the peptide backbone in the area of thesubstitution (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain.

The modified chondroitinase B has specific substitutions in specifiedportions of the peptide. In addition to these substitutions which may beconservative or non-conservative, other regions of the peptide mayinclude conservative substitutions that do not impact the activity ofthe modified chondroitinase B. One skilled in the art will appreciatethat the effect of a particular substitution can be evaluated by routinescreening assays, preferably the biological assays described herein.

According to the invention, isolated nucleic acid molecules that codefor a modified chondroitinase B polypeptide are provided and include:(a) nucleic acid molecules which hybridize under stringent conditions tothe nucleic acid equivalent which codes for a modified chondroitinase Bpolypeptide as described herein or parts thereof, (b) deletions,additions and substitutions of (a) which code for a respective modifiedchondroitinase B polypeptide or parts thereof, (c) nucleic acidmolecules that differ from the nucleic acid molecules of (a) or (b) incodon sequence due to the degeneracy of the genetic code, and (d)complements of (a), (b) or (c).

In certain embodiments, the nucleic acid molecule that codes for amodified chondroitinase B is highly homologous to the nucleic acidmolecules described herein. Preferably the homologous nucleic acidmolecule comprises a nucleotide sequence that is at least about 90%identical to the nucleotide sequence provided herein. More preferably,the nucleotide sequence is at least about 95% identical, at least about97% identical, at least about 98% identical, or at least about 99%identical to the nucleotide sequence provided herein. The homology canbe calculated using various, publicly available software tools wellknown to one of ordinary skill in the art. Exemplary tools include theBLAST system available from the website of the National Center forBiotechnology Information (NCBI) at the National Institutes of Health.

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

Optionally the modified chondroitinase B is recombinantly produced. Suchmolecules may be recombinantly produced using a vector including acoding sequence operably joined to one or more regulatory sequences. Asused herein, a coding sequence and regulatory sequences are said to be“operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein the codingsequences are operably joined to regulatory sequences. Two DNA sequencesare said to be operably joined if induction of a promoter in the 5′regulatory sequences results in the transcription of the coding sequenceand if the nature of the linkage between the two DNA sequences does not(1) result in the introduction of a frame-shift mutation, (2) interferewith the ability of the promoter region to direct the transcription ofthe coding sequences, or (3) interfere with the ability of thecorresponding RNA transcript to be translated into a protein. Thus, apromoter region would be operably joined to a coding sequence if thepromoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translatingsequences involved with initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Promoters may beconstitutive or inducible. Regulatory sequences may also includeenhancer sequences or upstream activator sequences, as desired.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmidsand phagemids. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium, or just a single time per host as thehost reproduces by mitosis. In the case of phage, replication may occuractively during a lytic phase or passively during a lysogenic phase. Anexpression vector is one into which a desired DNA sequence may beinserted by restriction and ligation such that it is operably joined toregulatory sequences and may be expressed as an RNA transcript. Vectorsmay further contain one or more marker sequences suitable for use in theidentification of cells which have or have not been transformed ortransfected with the vector. Markers include, for example, genesencoding proteins which increase or decrease either resistance orsensitivity to antibiotics or other compounds, genes which encodeenzymes whose activities are detectable by standard assays known in theart (e.g., B-galactosidase or alkaline phosphatase), and genes whichvisibly affect the phenotype of transformed or transfected cells, hosts,colonies or plaques. Preferred vectors are those capable of autonomousreplication and expression of the structural gene products present inthe DNA segments to which they are operably joined.

The term “high stringency conditions” as used herein refers toparameters with which the art is familiar. Nucleic acid hybridizationparameters may be found in references that compile such methods, e.g.Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. One example ofhigh-stringency conditions is hybridization at 65° C. in hybridizationbuffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% BovineSerum Albumin, 2.5 mM NaH₂PO₄(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15Msodium chloride/0.015M sodium citrate, pH7; SDS is sodium dodecylsulphate; and EDTA is ethylenediaminetetracetic acid. Afterhybridization, a membrane upon which the nucleic acid is transferred iswashed, for example, in 2×SSC at room temperature and then at0.1-0.5×SSC/0.1×SDS at temperatures up to 68° C. There are otherconditions, reagents, and so forth which can be used, which result inthe same degree of stringency. A skilled artisan will be familiar withsuch conditions, and thus they are not given here.

The skilled artisan also is familiar with the methodology for screeningcells for expression of such molecules, which then are routinelyisolated, followed by isolation of the pertinent nucleic acid. Thus,homologs and alleles of the modified chondroitinase B, as well asnucleic acids encoding the same, may be obtained routinely, and theinvention is not intended to be limited to the specific sequencesdisclosed.

For prokaryotic systems, plasmid vectors that contain replication sitesand control sequences derived from a species compatible with the hostmay be used. Examples of suitable plasmid vectors include pBR322, pUC18,pUC19 and the like; suitable phage or bacteriophage vectors includeλgt10, λgt11 and the like; and suitable virus vectors include pMAM-neo,pKRC and the like. Preferably, the selected vector of the presentinvention has the capacity to autonomously replicate in the selectedhost cell. Useful prokaryotic hosts include bacteria such as E. coli,Flavobacterium heparinum, Bacillus, Streptomyces, Pseudomonas,Salmonella, Serratia, and the like.

To express the modified chondroitinase B in a prokaryotic cell, it isdesirable to operably join the nucleic acid sequence of a modifiedchondroitinase B to a functional prokaryotic promoter. Such promoter maybe either constitutive or, more preferably, regulatable (i.e., inducibleor derepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, the bla promoter of the β-lactamase genesequence of pBR322, and the CAT promoter of the chloramphenicol acetyltransferase gene sequence of pPR325, and the like. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage λ (P₁ and P_(R)), the trp, recA, lacZ, lacI, and galpromoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol.162:176-182 (1985)) and the ζ-28-specific promoters of B. subtilis(Gilman et al., Gene sequence 32:11-20 (1984)), the promoters of thebacteriophages of Bacillus (Gryczan, In: The Molecular Biology of theBacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters(Ward et al., Mol. Gen. Genet. 203:468-478 (1986)).

Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol.1:277-282 (1987)); Cenatiempo (Biochimie 68:505-516 (1986)); andGottesman (Ann. Rev. Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the encoding sequence. Such ribosomebinding sites are disclosed, for example, by Gold et al. (Ann. Rev.Microbiol. 35:365-404 (1981)).

Because prokaryotic cells may not produce the modified chondroitinase Bwith normal eukaryotic glycosylation, expression of the modifiedchondroitinase B in eukaryotic hosts is useful when glycosylation isdesired. Preferred eukaryotic hosts include, for example, yeast, fungi,insect cells, and mammalian cells, either in vivo or in tissue culture.Mammalian cells which may be useful as hosts include HeLa cells, cellsof fibroblast origin such as VERO or CHO-K1, or cells of lymphoidorigin, such as the hybridoma SP2/0-AG14 or the myeloma P3×63Sg8, andtheir derivatives. Preferred mammalian host cells include SP2/0 andJ558L, as well as neuroblastoma cell lines such as IMR 332 that mayprovide better capacities for correct post-translational processing.Embryonic cells and mature cells of a transplantable organ also areuseful according to some aspects of the invention.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.

Another preferred host is an insect cell, for example in Drosophilalarvae. Using insect cells as hosts, the Drosophila alcoholdehydrogenase promoter can be used (Rubin, Science 240:1453-1459(1988)). Alternatively, baculovirus vectors can be engineered to expresslarge amounts of the modified chondroitinase B in insect cells (Jasny,Science 238:1653 (1987); Miller et al., In: Genetic Engineering (1986),Setlow, J. K., et al., eds., Plenum, Vol. 8, pp. 277-297).

Any of a series of yeast gene sequence expression systems whichincorporate promoter and termination elements from the genes coding forglycolytic enzymes and which are produced in large quantities when theyeast are grown in media rich in glucose may also be utilized. Knownglycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provide substantial advantages inthat they can also carry out post-translational peptide modifications. Anumber of recombinant DNA strategies exist which utilize strong promotersequences and high copy number plasmids which can be utilized forproduction of the desired proteins in yeast. Yeast recognize leadersequences on cloned mammalian gene sequence products and secretepeptides bearing leader sequences (i.e., pre-peptides).

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus, simian virus,or the like, where the regulatory signals are associated with aparticular gene sequence which has a high level of expression.Alternatively, promoters from mammalian expression products, such asactin, collagen, myosin, and the like, may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the gene sequences can bemodulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or which are subject to chemical (such asmetabolite) regulation.

The modified chondroitinase B is useful as an enzymatic tool due to itssubstrate specificity and specific activity and for cleavingpolysaccharides. The modified chondroitinase B may be used tospecifically cleave a polysaccharide by contacting the polysaccharidesubstrate with the modified chondroitinase B. The invention is useful ina variety of in vitro, in vivo and ex vivo methods in which it is usefulto cleave polysaccharides.

As used herein, a “polysaccharide” is a polymer composed ofmonosaccharides linked to one another. In many polysaccharides the basicbuilding block of the polysaccharide is actually a disaccharide unit,which can be repeating or non-repeating. Thus, a unit when used withrespect to a polysaccharide refers to a basic building block of apolysaccharide and can include a monomeric building block(monosaccharide) or a dimeric building block (disaccharide). The termpolysaccharide is also intended to embrace an oligosaccharide.Polysaccharides include but are not limited to glycosaminoglycans suchas chondroitin sulfate, dermatan sulfate, heparin, heparin-likeglycosaminoglycans (HLGAGs), heparan sulfate, hyaluronic acid, keratansulfate, and derivatives or analogs thereof, chitin in derivatives andanalogs thereof.

In addition to polysaccharides from natural sources, the polysaccharidesof the invention also include molecules that are biotechnologicallyprepared, chemically modified and synthetic. The term “biotechnologicalprepared” encompasses polysaccharides that are prepared from naturalsources of polysaccharides which have been chemically modified. This isdescribed for example in Razi et al., Bioche. J. 1995 Jul. 15;309 (Pt2): 465-72 and in Yates et al., Carbohydrate Res (1996) Nov. 20;294:15-27, and is known to those of skill in the art. Syntheticpolysaccharides are also well known to those of skill in the art and isdescribed in Petitou, M. et al., Bioorg Med Chem Lett. (1999) Apr.19;9(8):1161-6.

Analyses of polysaccharides as described in the present disclosure arepossible using modified chondroitinase B alone or in conjunction withother enzymes. Other polysaccharide degrading enzymes include but arenot limited to other chondroitinases (e.g. chondroitinase ABC andchondroitinase AC), hyaluronate lyase, heparinase-I, heparinase-II,heparinase-III, keratanase, D-glucuronidase and L-iduronidase, modifiedversions of these enzymes, variants and functionally active fragmentsthereof.

The methods that may be used to test the specific activity of modifiedchondroitinase B include those described in the Examples. The term“specific activity” as used herein refers to the enzymatic activity of apreparation of chondroitinase B. These methods may also be used toassess the function of variants and functionally active fragments ofmodified chondroitinase B. The k_(cat) value may be determined using anyenzymatic activity assay to assess the activity of a modifiedchondroitinase B enzyme. Several such assays are well-known in the art.For instance, an assay for measuring k_(cat) is described in (Ernst, S.E., Venkataraman, G., Winkler, S., Godavarti, R., Langer, R., Cooney, C.and Sasisekharan. R. (1996) Biochem. J. 315, 589-597). The “nativemodified chondroitinase B k_(cat) value” is the measure of enzymaticactivity of the native modified chondroitinase B obtained from celllysates of F. heparinum also described in the Examples below.

Due to the activity of modified chondroitinase B on polysaccharides, theproduct profile produced by a modified chondroitinase B may bedetermined by any method known in the art for examining the type orquantity of degradation product produced by modified chondroitinase Balone or in combination with other enzymes. One of skill in the art willalso recognize that the modified chondroitinase B may also be used toassess the purity of polysaccharides in a sample. One preferred methodfor determining the type and quantity of product is described inRhomberg, A. J. et al., PNAS, v. 95, p. 4176-4181 (April 1998), which ishereby incorporated in its entirety by reference. The method disclosedin the Rhomberg reference utilizes a combination of mass spectrometryand capillary electrophoretic techniques to identify the enzymaticproducts produced by heparinase. The Rhomberg study utilizes heparinaseto degrade HLGAGs to produce HLGAG oligosaccharides. MALDI(Matrix-Assisted Laser Desorption Ionization) mass spectrometry can beused for the identification and semiquantitative measurement ofsubstrates, enzymes, and end products in the enzymatic reaction. Thecapillary electrophoresis technique separates the products to resolveeven small differences amongst the products and is applied incombination with mass spectrometry to quantitate the products produced.Capillary electrophoresis may even resolve the difference between adisaccharide and its semicarbazone derivative.

The modified chondroitinase may also be used as a tool to sequencepolysaccharides. Detailed methods for sequencing polysaccharides andother polymers are disclosed in co-pending U.S. patent application Ser.No. 09/557,997 and Ser. No. 09/558,137, both filed on Apr. 24, 2000 andhaving common inventorship. The entire contents of both applications arehereby incorporated by reference. Briefly, the method is performed byenzymatic digestion, followed by mass spectrometry and capillaryelectrophoresis. The enzymatic assays can be performed in a variety ofmanners, as long as the assays are performed similarly on the modifiedchondroitinase B, so that the results may be compared. In the exampledescribed in the Rhomberg reference, enzymatic reactions are performedby adding 1 microliter of enzyme solution to 5 microliter of substratesolution. The digestion is then carried out at room temperature (22°C.), and the reaction is stopped at various time points by removing 0.5microliter of the reaction mixture and adding it to 4.5 microliter of aMALDI matrix solution, such as caffeic acid (approximately 12 mg/mL) and70% acetonitrile/water. The reaction mixture is then subjected to MALDImass spectrometry. The MALDI surface is prepared by the method of Xiangand Beavis (Xiang and Beavis (1994) Rapid. Commun. Mass. Spectrom. 8,199-204). A two-fold lower access of basic peptide (Arg/Gly)₁₅ ispremixed with matrix before being added to the oligosaccharide solution.A 1 microliter aliquot of sample/matrix mixture containing 1-3 picomolesof oligosaccharide is deposited on the surface. After crystallizationoccurs (typically within 60 seconds), excess liquid is rinsed off withwater. MALDI mass spectrometry spectra is then acquired in the linearmode by using a PerSeptive Biosystems (Framingham, Mass.) Voyager Elitereflectron time-of-flight instrument fitted with a 337 nanometernitrogen laser. Delayed extraction is used to increase resolution (22kV, grid at 93%, guidewire at 0.15%, pulse delay 150 ns, low mass gateat 1,000, 128 shots averaged). Mass spectra are calibrated externally byusing the signals for proteinated (Arg/Gly)₁₅ and its complex with theoligosaccharide.

Capillary electrophoresis may then be performed on aHewlett-Packard^(3D) CE unit by using uncoated fused silica capillaries(internal diameter 75 micrometers, outer diameter 363 micrometers,1_(det) 72.1 cm, and 1_(tot) 85 cm). Analytes are monitored by using UVdetection at 233 nm and an extended light path cell (Hewlett-Packard).The electrolyte is a solution of 10 microliter dextran sulfate and 50millimolar Tris/phosphoric acid (pH 2.5). Dextran sulfate is used tosuppress nonspecific interactions of the glycosaminoglycanoligosaccharides with a silica wall. Separations are carried out at 30kV with the anode at the detector side (reversed polarity). A mixture ofa 1/5-naphtalenedisulfonic acid and 2-naphtalenesulfonic acid (10micromolar each) is used as an internal standard.

Additionally, the coupling of CE and MALDI-MS with enzymes and abioinformatics-based, property-encoded nomenclature (PEN) have led to asequencing strategy (PEN-MALDI) described in (Venkataraman, G., Shriver,Z., Raman, R., and Sasisekharan, R. (1999) Science 286, 537-42).

Other methods for assessing the product profile may also be utilized.For instance, other methods include methods which rely on parameterssuch as viscosity (Jandik, K. A., Gu, K. and Linhardt, R. J., (1994),Glycobiology, 4:284-296) or total UV absorbance (Ernst, S. et al.,(1996), Biochem. J., 315:589-597) or mass spectrometry or capillaryelectrophoresis alone.

One of ordinary skill in the art, in light of the present disclosure, isenabled to produce preparations of glycosaminoglycan (GAG) fragmentcompositions utilizing the modified chondroitinase B molecules alone orin conjunction with other enzymes. These GAG fragments have manytherapeutic utilities. The GAG fragment preparations are prepared frompolysaccharide sources. A “polysaccharide source” as used herein refersto glycosaminoglycan composition which can be manipulated to produce GAGfragments. As described above, GAGs include but are not limited toisolated chondroitin sulfate, dermatan sulfate as well as chemicallymodified, biotechnology prepared and synthetic versions of suchpolysaccharides. Thus GAGs can be isolated from natural sources,prepared by direct synthesis.

The term “GAG fragment” as used herein refers to a GAG which hastherapeutic activity. For instance, the GAG fragment can prevent theproliferation and/or metastasis of a tumor cell. The use of the GAGfragments for other desired therapeutic activities are described below.Such compounds may be generated using modified chondroitinase B toproduce therapeutic fragments or they may be synthesized de novo basedon information derived from the use of modified chondroitinase B.Putative GAG fragments can be tested for therapeutic activity using anyof the assays described herein or known in the art. Thus the therapeuticGAG fragment may be a synthetic GAG fragment generated based on thesequence of the GAG fragment identified when a polysaccharide source iscontacted with modified chondroitinase B, or having minor variationswhich do not interfere with the activity of the compound. Alternativelythe therapeutic GAG fragment may be an isolated GAG fragment producedwhen the polysaccharide source is contacted with modified chondroitinaseB.

Thus, the methods of the invention enable one of skill in the art toprepare or identify an appropriate composition of GAG fragments,depending on the subject and the disorder being treated. Thesecompositions of GAG fragments may be used alone or in combination withthe modified chondroitinase B and/or other enzymes. Likewise modifiedchondroitinase B may also be used to produce GAG fragments in vivo.

The modified chondroitinase B molecules and/or GAG fragments producedusing the modified chondroitinase B can be used for the treatment of anytype of condition in which chondroitinase therapy or GAG fragmenttherapy has been identified as a useful therapy, e.g., preventingcoagulation, inhibiting angiogenesis, inhibiting proliferation. Themodified chondroitinase B and/or GAG fragments can also be used formediating cell signaling. Thus, the invention is useful in a variety ofin vitro, in vivo and ex vivo methods in which therapies are useful. Forinstance, it is known that GAG fragments and chondroitinase B are usefulfor preventing coagulation, inhibiting cancer cell growth andmetastasis, preventing angiogenesis, preventing neovascularization,preventing psoriasis. Chondroitinase B is also useful in the treatmentof ostoearthritis and maternal malarial infection. The GAG fragmentcompositions may also be used in in vitro assays, such as a qualitycontrol sample.

Each of these disorders is known in the art and is described, forinstance, in Harrison's Principles of Internal Medicine (McGraw Hill,Inc., New York), which is incorporated by reference.

In one embodiment the preparations of the invention are used forinhibiting angiogenesis. An effective amount for inhibiting angiogenesisof the GAG fragment preparation or modified chondroitinase B isadministered to a subject in need of treatment thereof. Angiogenesis asused herein is the inappropriate formation of new blood vessels.“Angiogenesis” often occurs in tumors when endothelial cells secrete agroup of growth factors that are mitogenic for endothelium causing theelongation and proliferation of endothelial cells which results in ageneration of new blood vessels. Several of the angiogenic mitogens areheparin binding peptides which are related to endothelial cell growthfactors. The inhibition of angiogenesis can cause tumor regression inanimal models, suggesting a use as a therapeutic anticancer agent. Aneffective amount for inhibiting angiogenesis is an amount of GAGfragment preparation or a modified chondroitinase B which is sufficientto diminish the number of blood vessels growing into a tumor. Thisamount can be assessed in an animal model of tumors and angiogenesis,many of which are known in the art.

The modified chondroitinase B molecules and GAG fragment preparation areuseful for treating or preventing disorders associated with coagulation.A “disease associated with coagulation” as used herein refers to acondition characterized by inflammation resulting from an interruptionin the blood supply to a tissue, which may occur due to a blockage ofthe blood vessel responsible for supplying blood to the tissue such asis seen for myocardial, cerebral infarction, or peripheral vasculardisease, or as a result of embolism formation associated with conditionssuch as atrial fibrillation or deep venous thrombosis. A cerebralischemic attack or cerebral ischemia is a form of ischemic condition inwhich the blood supply to the brain is blocked. This interruption in theblood supply to the brain may result from a variety of causes, includingan intrinsic blockage or occlusion of the blood vessel itself, aremotely originated source of occlusion, decreased perfusion pressure orincreased blood viscosity resulting in inadequate cerebral blood flow,or a ruptured blood vessel in the subarachnoid space or intracerebraltissue.

The modified chondroitinase B or the GAG fragments generated therewithmay be used alone or in combination with a therapeutic agent fortreating a disease associated with coagulation. Examples of therapeuticsuseful in the treatment of diseases associated with coagulation includeanticoagulation agents, antiplatelet agents, and thrombolytic agents.

Anticoagulants include, but are not limited to, heparin, warfarin,coumadin, dicumarol, phenprocoumon, acenocoumarol, ethyl biscoumacetate,and indandione derivatives.

Antiplatelet agents include, but are not limited to, aspirin,thienopyridine derivatives such as ticlopodine and clopidogrel,dipyridamole and sulfinpyrazone, as well as RGD mimetics and alsoantithrombin agents such as, but not limited to, hirudin.

Thrombolytic agents include, but are not limited to, plasminogen,a₂-antiplasmin, streptokinase, antistreplase, tissue plasminogenactivator (tPA), and urokinase.

The invention also encompasses screening assays for identifyingtherapeutic GAG fragments for the treatment of a tumor and forpreventing metastasis. The assays may be accomplished by treating atumor or isolated tumor cells with modified chondroitinase B and/orother native or modified heparinases and isolating the resultant GAGfragments. The isolated GAG fragments may then be tested for therapeuticactivity in the prevention of tumor cell proliferation and metastasis.Thus the invention encompasses individualized therapies, in which atumor or portion of a tumor is isolated from a subject and used toprepare the therapeutic GAG fragments. These therapeutic fragments canbe re-administered to the subject to protect the subject from furthertumor cell proliferation or metastasis or from the initiation ofmetastasis if the tumor is not yet metastatic. Alternatively thefragments can be used in a different subject having the same type ortumor or a different type of tumor.

The compositions of the invention are useful for treating and preventingcancer cell proliferation and metastasis. Thus, according to anotheraspect of the invention, there is provided methods for treating subjectshaving or at risk of having cancer. The terms “treat” and “treating”tumor cell proliferation as used herein refer to inhibiting completelyor partially the proliferation or metastasis of a cancer or tumor cell,as well as inhibiting any increase in the proliferation or metastasis ofa cancer or tumor cell.

A “subject having a cancer” is a subject that has detectable cancerouscells. The cancer may be a malignant or non-malignant cancer. Cancers ortumors include but are not limited to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas.

A “subject at risk of having a cancer” as used herein is a subject whohas a high probability of developing cancer. These subjects include, forinstance, subjects having a genetic abnormality, the presence of whichhas been demonstrated to have a correlative relation to a higherlikelihood of developing a cancer and subjects exposed to cancer causingagents such as tobacco, asbestos, or other chemical toxins, or a subjectwho has previously been treated for cancer and is in apparent remission.When a subject at risk of developing a cancer is treated with a modifiedchondroitinase B or degradation product thereof the subject may be ableto kill the cancer cells as they develop.

Effective amounts of the modified chondroitinase B, or GAG fragments ofthe invention are administered to subjects in need of such treatment.Effective amounts are those amounts which will result in a desiredimprovement in the condition or symptoms of the condition, e.g., forcancer this is a reduction in cellular proliferation or metastasis. Suchamounts can be determined with no more than routine experimentation. Itis believed that doses ranging from 1 nanogram/kilogram to 100milligrams/kilogram, depending upon the mode of administration, will beeffective. The absolute amount will depend upon a variety of factors(including whether the administration is in conjunction with othermethods of treatment, the number of doses and individual patientparameters including age, physical condition, size and weight) and canbe determined with routine experimentation. It is preferred generallythat a maximum dose be used, that is, the highest safe dose according tosound medical judgment. The mode of administration may be any medicallyacceptable mode including oral, subcutaneous, intravenous, etc.

In some aspects of the invention the effective amount of modifiedchondroitinase B or GAG fragment is that amount effective to preventinvasion of a tumor cell across a barrier. The invasion and metastasisof cancer is a complex process which involves changes in cell adhesionproperties which allow a transformed cell to invade and migrate throughthe extracellular matrix (ECM) and acquire anchorage-independent growthproperties. Liotta, L. A., et al., Cell 64:327-336 (1991). Some of thesechanges occur at focal adhesions, which are cell/ECM contact pointscontaining membrane-associated, cytoskeletal, and intracellularsignaling molecules. Metastatic disease occurs when the disseminatedfoci of tumor cells seed a tissue which supports their growth andpropagation, and this secondary spread of tumor cells is responsible forthe morbidity and mortality associated with the majority of cancers.Thus the term “metastasis” as used herein refers to the invasion andmigration of tumor cells away from the primary tumor site.

The barrier for the tumor cells may be an artificial barrier in vitro ora natural barrier in vivo. In vitro barriers include but are not limitedto extracellular matrix coated membranes, such as Matrigel. Thus themodified chondroitinase B compositions or degradation products thereofcan be tested for their ability to inhibit tumor cell invasion in aMatrigel invasion assay system as described in detail by Parish, C. R.,et al., “A Basement-Membrane Permeability Assay which Correlates withthe Metastatic Potential of Tumour Cells,” Int. J. Cancer (1992)52:378-383. Matrigel is a reconstituted basement membrane containingtype IV collagen, laminin, heparan sulfate proteoglycans such asperlecan, which bind to and localize bFGF, vitronectin as well astransforming growth factor-β (TGF-β), urokinase-type plasminogenactivator (uPA), tissue plasminogen activator (tPA), and the serpinknown as plasminogen activator inhibitor type 1 (PAI-1). Other in vitroand in vivo assays for metastasis have been described in the prior art,see, e.g., U.S. Pat. No. 5,935,850, issued on Aug. 10, 1999, which isincorporated by reference. An in vivo barrier refers to a cellularbarrier present in the body of a subject.

When administered to a patient undergoing cancer treatment, the modifiedchondroitinase B or GAG fragment may be administered in cocktailscontaining other anti-cancer agents. The compounds may also beadministered in cocktails containing agents that treat the side-effectsof radiation therapy, such as anti-emetics, radiation protectants, etc.

The modified chondroitinase B or GAG compounds may also be linked to atargeting molecule. A targeting molecule is any molecule or compoundwhich is specific for a particular cell or tissue and which can be usedto direct the modified chondroitinase B or GAG to the cell or tissue.Preferably the targeting molecule is a molecule which specificallyinteracts with a cancer cell or a tumor. For instance, the targetingmolecule may be a protein or other type of molecule that recognizes andspecifically interacts with a tumor antigen.

The preparations of the present invention may also be used to inhibitbinding to CS/DS proteoglycans that act as cell adhesion molecules,particularly during infection (e.g. malarial infection). It has beenfound that in pregnant women infected with Plasmodium falciparuminfected red blood cells (IRBCs) accumulate in the placenta. Theaccumulation of IRBCs is believed to be due to the adhesion of IRBCmembrane proteins to molecules found in the intervillous space in theplacenta such as chondroitin 4-sulfate (Achur et. al., 2000, The Journalof Biological Chemistry, Vol. 275, No. 51 and Alkhalil, et. al., 2000,The Journal of Biological Chemistry, Vol. 275, No. 51). One aspect ofthe present invention, therefore, is a method for inhibiting maternalmalarial infection. An effective amount for treating malarial infectionis that amount that leads to a decrease in the number of infected redblood cells in the placenta sufficient that eliminate or decrease theundesirable effects of malarial infection during pregnancy. Theseeffects include: low birth weight, still birth, abortion, prematuredelivery and maternal morbidity and mortality (Achur et. al., 2000, TheJournal of Biological Chemistry, Vol. 275, No. 51).

The modified chondroitinase B is, in some embodiments, immobilized on asupport. The modified chondroitinase B may be immobilized to any type ofsupport but if the support is to be used in vivo or ex vivo it isdesired that the support is sterile and biocompatible. A biocompatiblesupport is one which would not cause an immune or other type of damagingreaction when used in a subject. The modified chondroitinase B may beimmobilized by any method known in the art. Many methods are known forimmobilizing proteins to supports. A “solid support” as used hereinrefers to any solid material to which a polypeptide can be immobilized.

Solid supports, for example, include but are not limited to membranes,e.g., natural and modified celluloses such as nitrocellulose or nylon,Sepharose, Agarose, glass, polystyrene, polypropylene, polyethylene,dextran, amylases, polyacrylamides, polyvinylidene difluoride, otheragaroses, and magnetite, including magnetic beads. The carrier can betotally insoluble or partially soluble and may have any possiblestructural configuration. Thus, the support may be spherical, as in abead, or cylindrical, as in the inside surface of a test tube ormicroplate well, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, bottom surface of amicroplate well, etc.

The modified chondroitinase B may also be used to remove active GAGsfrom a GAG containing fluid. A GAG containing fluid is contacted withthe modified chondroitinase B of the invention to degrade the GAG. Themethod is particularly useful for the ex vivo removal of GAGs fromblood. In one embodiment the modified chondroitinase B may beimmobilized on a solid support as is conventional in the art. The solidsupport containing the immobilized modified chondroitinase B may be usedin extracorporeal medical devices (e.g. hemodialyzer, pump-oxygenator)to prevent the blood in the device from clotting. The support membranecontaining immobilized modified chondroitinase B is positioned at theend of the device to neutralize the GAG before the blood is returned tothe body.

In general, when administered for therapeutic purposes, the formulationsof the invention are applied in pharmaceutically acceptable solutions.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, adjuvants, and optionally other therapeutic ingredients.

The compositions of the invention may be administered per se (neat) orin the form of a pharmaceutically acceptable salt. When used in medicinethe salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts of the carboxylic acidgroup.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V);citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V);and phosphoric acid and a salt (0.8-2% W/V). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9%W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).

The present invention provides pharmaceutical compositions, for medicaluse, which comprise modified chondroitinase B and/or GAG fragmentstogether with one or more pharmaceutically acceptable carriers andoptionally other therapeutic ingredients. The term“pharmaceutically-acceptable carrier” as used herein, and described morefully below, means one or more compatible solid or liquid filler,dilutants or encapsulating substances which are suitable foradministration to a human or other animal. In the present invention, theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the modified chondroitinase B orGAG fragments, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular active agentselected, the particular condition being treated and the dosage requiredfor therapeutic efficacy. The methods of this invention, generallyspeaking, may be practiced using any mode of administration that ismedically acceptable, meaning any mode that produces effective levels ofan immune response without causing clinically unacceptable adverseeffects. A preferred mode of administration is a parenteral route. Theterm “parenteral” includes subcutaneous injections, intravenous,intramuscular, intraperitoneal, intrasternal injection or infusiontechniques. Other modes of administration include oral, mucosal, rectal,vaginal, sublingual, intranasal, intratracheal, inhalation, ocular,transdermal, etc.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polylactic andpolyglycolic acid, polyanhydrides and polycaprolactone; nonpolymersystems that are lipids including sterols such as cholesterol,cholesterol esters and fatty acids or neutral fats such as mono-, di andtriglycerides; hydrogel release systems; silastic systems; peptide basedsystems; wax coatings, compressed tablets using conventional binders andexcipients, partially fused implants and the like. Specific examplesinclude, but are not limited to: (a) erosional systems in which thepolysaccharide is contained in a form within a matrix, found in U.S.Pat. No. 4,452,775 (Kent); U.S. Pat. No. 4,667,014 (Nestor et al.); andU.S. Pat. Nos. 4,748,034 and 5,239,660 (Leonard) and (b) diffusionalsystems in which an active component permeates at a controlled ratethrough a polymer, found in U.S. Pat. No. 3,832,253 (Higuchi et al.) andU.S. Pat. No. 3,854,480 (Zaffaroni). In addition, a pump-based hardwaredelivery system can be used, some of which are adapted for implantation.

Controlled release of modified chondroitinase B or GAG fragments canalso be achieved with appropriate excipient materials that arebiocompatible and biodegradable. These polymeric materials which effectslow release of the modified chondroitinase B or GAG fragments may beany suitable polymeric material for generating particles, including, butnot limited to, nonbioerodable/non-biodegradable andbioerodable/biodegradable polymers. Such polymers have been described ingreat detail in the prior art. They include, but are not limited to:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, polyvinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andcopolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulfate sodium salt, poly (methylmethacrylate), poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexlmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinylchloride polystyrene, polyvinylpryrrolidone, hyaluronic acid, andchondroitin sulfate.

Examples of preferred non-biodegradable polymers include ethylene vinylacetate, poly(meth) acrylic acid, polyamides, copolymers and mixturesthereof.

Examples of preferred biodegradable polymers include synthetic polymerssuch as polymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),poly(caprolactone), poly(hydroxybutyrate), poly(lactide-co-glycolide)and poly(lactide-co-caprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion. The foregoing materials may be usedalone, as physical mixtures (blends), or as co-polymers. The mostpreferred polymers are polyesters, polyanhydrides, polystyrenes andblends thereof.

A subject is any human or non-human vertebrate, e.g., dog, cat, horse,cow, pig.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

Materials and Methods

Materials

Porcine intestinal mucosa dermatan sulfate, chondroitin 4-sulfate, andchondroitin 6-sulfate were purchased from Sigma (St. Louis, Mo.). Thedisaccharide standards were from Seikagaku/Associates of Cape Cod(Falmouth, Mass.). Oligonucleotide primers for PCR mutagenesis were fromInvitrogen (Carlsbad, Calif.).

Docking of Dermatan Sulfate Tetrasaccharide into Chondroitinase B ActiveSite

The structure of the dermatan sulfate tetrasaccharide was obtained froma recently solved co-crystal structure of a chondroitinase AC mutantenzyme with a dermatan sulfate hexasaccharide (PDB id: 1HM2). Only fourof the sugar units in this hexasaccharide were defined in the co-crystalstructure (Huang, W., Boju, L., Tkalec, L., Su, H., Yang, H. O., Gunay,N. S., Linhardt, R. J., Kim, Y. S., Matte, A., and Cygler, M. (2001)Biochemistry 40, 2359-72). Therefore, we used the definedtetrasaccharide region, ΔUA(1→3)GalNAc4S(1→4)IdoA(1→3)GalNAc4S, in ourdocking study. The initial orientation of this dermatan sulfatestructure relative to chondroitinase B was obtained by superimposing thenon-reducing end of the tetrasaccharide with the disaccharide in theco-crystal structure. This preliminary orientation was modified bymanually manipulating the tetrasaccharide structure to optimizefavorable contacts between the active site amino acids and thetetrasaccharide. All the manipulations of the structures and dockingwere done using the Viewer and Docking module of INSIGHTII.

The manually modified docked tetrasaccharide was subjected to a energyminimization process in which the potentials of the enzyme and theoligosaccharide were set using the AMBER force field modified to includecarbohydrates (Homans, S. W. (1990) Biochemistry 29, 9110-8) withsulfate and sulfamate groups (Huige, C. J. M., and Altona, C. (1995) J.Comp. Chem. 16, 56-7926). The enzyme-substrate complex was subjected to300 steps of steepest gradient minimization without including charges,keeping most of the enzyme fixed, and allowing only the regions close tothe substrate to move. A force constant of 5,000 kcal was applied toeach of the ring torsion angles ensuring that the ring geometries of thesugar units in the tetrasaccharide were not significantly distorted.Each of the subsequent orientations of the tetrasaccharide substrate wasevaluated for steric contacts and non-bonded interactions with theactive site of the enzyme. The optimal orientation with reasonably lowsteric hindrance was selected for further energy minimization. Therefined structure was further subjected to 300 steps of conjugategradient minimization including charges. A distance-dependent dielectricwith a scaling factor of 4.0 and 1-4 nonbonded scaling factor of 0.5were set while using AMBER force field as recommended by the softwaremanual.

PCR Site-Directed Mutagenesis of Chondroitinase B

Lys250, Arg271, His272, Glu333, Arg363, and Arg364 were mutated toalanine using overlap extension PCR for 15 cycles (Pojasek, K., Shriver,Z., Hu, Y., and Sasisekharan, R. (2000) Biochemistry 39, 4012-9). Theprimer sequences used for each of the mutants are as follows:

H272A 5′: AAC TTT CGT GCC GGT GAT CAT (SEQ ID NO: 3) H272A 3′: ATG ATCACC GGC ACG AAA GTT (SEQ ID NO: 4) E333A 5′: ATG GCT TCG GCG CAT GCT CTT(SEQ ID NO: 5) E333A 3′: AAG AGC ATG CGC CGA AGC CAT (SEQ ID NO: 6)K250A 5′: ATC ACC AGC GCG TCG CAG GAA (SEQ ID NO: 7) K250A 3′: TTC CTGCGA AGC GCT GGT GAT (SEQ ID NO: 8) R271A 5′: ATG AAC TTT GCT CAC GGT GAT(SEQ ID NO: 9) R271A 3′: ATC ACC GTG AGC AAA GTT CAT (SEQ ID NO: 10)R363A 5′: TTG GAT GAG GCC AGA AAA GAA (SEQ ID NO: 11) R363A 3′: TTC TTTTCT GGC CTC ATC CAA (SEQ ID NO: 12) R364A 5′: GAT GAG CGC GCA AAA GAATAT (SEQ ID NO: 13) R364A 3′: ATA TTC TTT TGC GCG CTC ATC (SEQ ID NO:14)

The PCR reaction products were separated on an agarose gel and the bandcorresponding to the proper length was excised. The DNA was extractedfrom the gel using a Gel Purification Kit (Qiagen, Valencia, Calif.),the insert was subcloned into pCRT7/NT (Invitrogen, Carlsbad, Calif.),and the plasmid was prepared using a Miniprep kit (Qiagen). Each of theclones was sequenced to verify the presence of the individual alaninepoint mutations. Each chondroitinase B mutant was excised from pCRT7/NTusing Nde I and BamH I (New England Biolabs, Beverly, Mass.) enzymecocktail and subcloned into a pET15b expression vector (Novagen,Madison, Wis.) that had been digested previously with these sameenzymes. Recombinant chondroitinase B that had been cloned in a similarfashion was also expressed and compared to each of the alanine mutants.

Protein Expression and Purification

Recombinant chondroitinase B and the site-directed mutants wereexpressed and purified as previously described (Pojasek, K., Shriver,Z., Kiley, P., Venkataraman, G., and Sasisekharan, R. (2001) Biochem.Biophys. Res. Commun. 286, 343-51). Purity of recombinant chondroitinaseB and the site-directed mutants were assessed by SDS-polyacrylamide gelelectrophoresis analysis using precast 12% gels, the Mini-Protean IIapparatus, and the Silver Stain Plus kit (Bio-Rad, Hercules, Calif.). Arelative protein concentration was calculated using the Bradford Assay(Bio-Rad, Hercules, Calif.) with bovine serum albumin as a standard.

Kinetic Analysis

The activity of chondroitinase B and the various site-directed mutantswas determined by adding 10-50 μl of the sample to a 1 ml cuvettecontaining 1 mg/ml of dermatan sulfate in 50 mM Tris-HCl, pH 8.0 at 30°C. Product formation was monitored as an increase in absorbance at 232nm as a function of time (Pojasek, K., Shriver, Z., Kiley, P.,Venkataraman, G., and Sasisekharan, R. (2001) Biochem. Biophys. Res.Commun. 286, 343-51).

The kinetic parameters, K_(m) and k_(cat), were calculated forchondroitinase B and the site-directed mutants by obtaining the initialreaction rate (v_(o)) as a function of substrate concentration.Approximately 1 μg (13 pmol) of enzyme was added to a 1 ml of dermatansulfate at concentrations ranging from 0.010 μg/ml to 2 mg/ml. Theinitial rate was measured for 4-10 s at 30° C. in the same Tris-HClbuffer used for the activity assay. The slope of the resulting line,assuming zero order kinetics, was plotted versus the substrateconcentration using SigmaPlot (SSPS, Inc., Chicago, Ill.). The K_(m)(μM) and V_(max)(μM/s) were calculated using the Michaelis-Mentenequation: v₀=(V_(max)*[S])/(K_(m)+[S]). The k_(cat) (s⁻¹) was calculatedby dividing the V_(max) by the concentration of enzyme in the reaction.

Dermatan Sulfate Digestion and Capillary Electrophoresis

To examine changes in product profile of each site-directed mutant whencompared to recombinant chondroitinase B (20 μg), digests of 1 mg/mldermatan sulfate 50 mM Tris-HCl, pH 8.0 were performed for 12-14 hr. at30° C. The digests were analyzed using capillary electrophoresis aspreviously described (Pojasek, K., Shriver, Z., Kiley, P., Venkataraman,G., and Sasisekharan, R. (2001) Biochem. Biophys. Res. Commun. 286,343-51). Briefly, the chondroitinase B and site-directed mutant digestswere diluted twofold and analyzed with an extended path-length cell anda voltage of 30 kV applied using reverse polarity. The running bufferconsisted of 50 mM Tris, 10 μM dextran sulfate that had been brought toa pH of 2.5 using phosphoric acid and the saccharide products weredetected by monitoring at 232 nm.

The total peak area for the recombinant chondroitinase B and mutantdigest profiles was calculated by totaling the areas of theΔUA-GalNAc2S,4S; ΔUA-GalNAc4S,6S; and ΔUA-GalNAc4S peaks. The total peakarea for the R364A mutant also included the sum of the area of the threeadditional oligosaccharide peaks. The ratio of the ΔUA-GalNAc4S peakarea to the total peak area was then calculated for the recombinantchondroitinase B and each mutant for a comparison of overall enzymaticactivity.

MALDI Mass Spectrometry

The reaction products from the R364A digest of dermatan sulfate wereanalyzed using MALDI-MS. Samples were prepared using the basic peptide(RG)₁₅R as previously described (Rhomberg, A. J., Ernst, S.,Sasisekharan, R., and Biemann, K. (1998) Proc. Natl. Acad. Sci. USA 95,4176-81). MALDI-MS spectra were acquired on a Voyager Elite system(PerSeptive Biosystems, Framingham, Mass.) in the linear mode withdelayed extraction and similar instrument parameters to those describedpreviously (Rhomberg, A. J., Ernst, S., Sasisekharan, R., and Biemann,K. (1998) Proc. Natl. Acad. Sci. USA 95, 4176-81).

Circular Dichroism

Recombinantly expressed chondroitinase B and the inactive K250A mutantwere concentrated and buffer-exchanged into 50 mM sodium phosphate, pH7.0 using a Centricon 10 Filter (Millipore, Watertown, Mass.). CDspectra were collected on an Aviv 62DS spectropolarimeter equipped witha thermostatic temperature controller and interfaced to an IBMmicrocomputer. Measurements were performed in a quartz cell with a 1 mmpath length. Spectra were recorded at 25° C., in an average of 10 scansbetween 205 and 270 nm, with a 1.0 nm bandwidth and a scan rate of 3nm/min. CD band intensities are expressed as molar ellipticities, θ_(M),in degrees·cm²·dmol⁻¹.

Results and Discussion

Interactions between Chondroitinase B and Dermatan Sulfate Substrate

The structure of a previously crystallized DS tetrasaccharide was dockedinto the chondroitinase B active site. The direction of thetetrasaccharide relative to the enzyme was the same as the ΔUA-GalNAc4Sdisaccharide product in the co-crystal structure with the non-reducingend of the tetrasaccharide towards the C-terminus and the reducing endtowards the N-terminus of the enzyme. However, the orientation of thetetrasaccharide relative to the parallel beta-helical axis of the enzymewas different from that of the disaccharide (FIG. 1, (A)). When thenon-reducing end of the tetrasaccharide was superimposed with thedisaccharide product from the co-crystal structure, the orientation ofthe tetrasaccharide was such that its reducing end collided with a wallof the active site cleft (FIG. 1, (A)). Also, in this orientation, itsreducing end was too far apart from the basic cluster of residuesHis116, Arg184, and Arg218. Our docking energy minimization resulted inrepositioning of the tetrasaccharide substrate to achieve maximumcontact with the active site cleft of the enzyme (FIG. 1, (A)). In thefinal orientation, the tetrasaccharide completely occupied the −2, −1,+1, and +2 subsites (standard nomenclature) of the active site ofchondroitinase B.

Active Site Residues

The docked tetrasaccharide occupied all of the chondroitinase subsites,and the theoretical enzyme-substrate complex provided a better pictureof the interaction between the DS substrate and the active siteresidues. Glu333, Lys250, Arg271 and His 272 were identified as keyresidues involved in catalysis based on the proximity to the −1 and +1subsites containing cleavable −GalNAc4S-IdoA-linkage (FIG. 1, (B)). Thiscluster of charged residues in the catalytic site suggests that theremay be more than the prototypical triad of residues that are involved inthe proton abstraction and donation mechanism resulting in theβ-eliminative cleavage. Glu333 is positioned proximal to the O1 ofGalNAc4S in such a way that it could potentially mediate protonabstraction via a water molecule. The proximity of His272 and Lys250 tothe C5 proton (FIG. 1, (B)) indicates that these residues are alsopositioned to act as general base for proton abstraction. However,Lys250 is the only residue in proximity to the carboxylate moiety of theIdoA monosaccharide and supports its involvement in neutralizing thecharge of the carboxylate group. Arg271 is proximal to both the ringoxygen and O1 of GalNAc residue and thus is positioned to protonate theleaving O1 atom of the GalNAc after cleavage.

Substrate Binding Residues

Several residues involved in substrate binding were identified from ourtheoretical chondroitinase B-tetrasaccharide complex. These includebasic residues Arg318, Arg363 and Arg364 and pyranose ring stackingaromatic residues Phe296 and Trp298. Phe296 provides a parallel stackinginteraction with the IdoA in the −2 subsite and Trp298 stacksperpendicularly with the IdoA and GalNAc in subsite −2 and −1,respectively (FIG. 1, (B)). Arg364 is positioned to interact with boththe 4-O sulfate of the GalNAc4S and the carboxyl group of thenon-reducing end IdoA (FIG. 1, (B)). Since the 4-O sulfate group ofGalNAc4S and IdoA are hallmark modifications of dermatan sulfate, Arg364residue is most likely to be involved in substrate specificity of theenzyme. Arg318 interacts with the IdoA in the −2 site and Arg363 ispositioned to interact with an additional GalNAc4S moiety on thenonreducing end in what would potentially be subsite −3. Finally, Asn213interacts with the N-acetyl group of the GalNAc in the −1 subsite (FIG.1, (B)).

In the product release site (subsites +1 and +2), the side chains ofArg184 and His116 are oriented to provide favorable ionic interactionswith the GalNAc4S residue at the reducing end of the DS tetrasaccharide(FIG. 1, (B)). These interactions provide a more definitive meaning tothe speculated role of these two basic residues in binding to 4-Osulfate group at the reducing end of the DS substrate. Taken together,our enzyme-substrate complex provides a clear framework of the variousresidues involved in substrate binding and product release.

Active Site Symmetry

In addition to providing further insight into the exact role of eachresidue in the chondroitinase B active site, our conformational studyalso uncovered a chemical symmetry of amino acid side chains in thisregion. In fact, there appears to be an internal twofold symmetry of thepositively charged, negatively charged, and hydrophobic residues in theactive site about an axis passing through the cleavage site (−1 and +1)and perpendicular to the axis of the β helix (FIG. 2). Specifically theproposed residues that are involved in the substrate binding site (−2and −1), including Phe296, Arg318, Arg364, seem to have correspondingresidues in the product release site (+1 and +2), including Tyr222,Arg184 and Arg219 that are related by this symmetry. In addition, Glu245is in proximity of the catalytic site and appears to be related to theGlu333 residue by the same twofold symmetry (FIG. 2).

Understanding the significance of the active site symmetry providesvaluable insights into the mechanism by which chondroitinase Bdepolymerizes its DS substrate. Without being bound by any particulartheory, several plausible explanations regarding the importance of thisactive site symmetry are proposed. To begin with, the distance betweenthe carbonyl oxygens of both Glu245 and Glu333 is about 9.5 Å, adistance comparable to the diameter of the structure of the DS substrateprojected along the helical axis. Thus, if both of these negativelycharged glutamates are involved in catalysis, their symmetricalarrangement would facilitate the translation of the substrate throughthe active site cleft without the need for its rotation, leading to moreefficient DS depolymerization. In addition, this active site symmetrymay be involved in accommodating the perturbations in the DS chaincaused by the conformational flexibility of iduronic acid, a commoncomponent of dermatan sulfate (Venkataraman, G., Sasisekharan, V.,Cooney, C. L., Langer, R., and Sasisekharan, R. (1994) Proc. Natl. Acad.Sci. USA 91, 6171-5).

The symmetry of the active site may also be involved in defining thedirection that the substrate is processed through the active site.Interestingly, the DS-derived disaccharide in the co-crystal structurethat is an actual product of chondroitinase B action is in the substratebinding site, not the product release site. This observation, coupledwith the active site symmetry, suggests that the directionality of theactive site might be more complex than originally thought. In fact, thereducing end of a genuine substrate may be potentially oriented towardsthe C-terminal end of enzyme, a pattern of binding common among otherpolysaccharide lyases (Steinbacher, S., Seckler, R., Miller, S., Steipe,B., Huber, R., and Reinemer, P. (1994) Science 265, 383-6 and Scavetta,R. D., Herron, S. R., Hotchkiss, A. T., Kita, N., Keen, N. T., Benen, J.A., Kester, H. C., Visser, J., and Jurnak, F. (1999) Plant Cell 11,1081-92), and not towards the N-terminal end as seen in the co-crystalstructure (Huang, W., Matte, A., Li, Y., Kim, Y. S., Linhardt, R. J.,Su, H., and Cygler, M. (1999) J. Mol. Biol. 294, 1257-69). Thedirectionality of substrate binding within the active site ofpolysaccharide lyases is usually unambiguously defined by a structuralfeature similar to the presence of a Ca²⁺ ion at one end of the cleft asis the case with pectate lyase C from Erwinia chrysanthemi (Scavetta, R.D., Herron, S. R., Hotchkiss, A. T., Kita, N., Keen, N. T., Benen, J.A., Kester, H. C., Visser, J., and Jurnak, F. (1999) Plant Cell 11,1081-92). This underscores the uniqueness of the chondroitinase B activesite symmetry.

Mutagenesis and Active Site Characterization

Having identified the key substrate binding and catalytic residues usingour theoretical enzyme-substrate complex, we sought to establish theirfunctional roles using site-directed mutagenesis. The basic residues,Lys250, Arg271, and His272, were chosen based on their location in theactive site of chondroitinase B. In addition, the acidic residue, Glu333was chosen because of its possible role in proton abstraction. We alsomutated two of the residues implicated in substrate binding andspecificity, namely Arg363 and Arg364 to alanine. These site-directedmutants were cloned into pET15b and expressed along side the recombinantchondroitinase B.

Both H272A and E333A showed altered kinetics when compared with therecombinant chondroitinase B (Table 1). For instance, the K_(m) andk_(cat) for the H272A chondroitinase B mutant are 2.7 μM and 29 s⁻¹,respectively, compared to a K_(m) of 4.6 μM and a k_(cat) of 190 s⁻¹ forthe recombinant enzyme (Pojasek, K. et al. (2001) Biochem. Biophys. Res.Commun. 286, 343-51). The E333A mutant had similar alterations in K_(m)and k_(cat) (Table 1). Both of these mutations lead to a slightreduction in K_(m) while drastically reducing k_(cat). In fact, whencompared to the recombinant chondroitinase B, the H272A and the E333Amutants have a fourfold and a 26-fold decrease in k_(cat)/K_(m),respectively (Table 1).

TABLE 1 Kinetic Analysis of Chondroitinase B and Mutants KineticParameters^(a) Enzyme K_(m) (μM) k_(cat) (s⁻¹) k_(cat)/K_(m) (μM s⁻¹)Chondroitinase B 4.6 ± 0.31 190 ± 80  41 K250A n.d.^(b) n.d.^(b)n.d.^(b) H272A 2.7 ± 0.24 12 29 ± 3.0  11 E333A 2.8 ± 0.64 4.6 ± 1.6 1.6R363A 4.6 ± 0.49 404 ± 156 88 R364A n.d.^(b) n.d.^(b) n.d.^(b)^(a)Values are the mean of 3 experiments ± S.E. ^(b)Kinetics wereundetectable due to low activity of the mutant enzyme.

In addition to kinetic analysis, each of the mutant enzymes and therecombinant chondroitinase B were allowed to exhaustively digestdermatan sulfate to determine changes in product profile that may beliealterations in substrate specificity. These digests were diluted andanalyzed using capillary electrophoresis. Complete digestion of thedermatan substrate was seen with the chondroitinase B reaction asindicated by a major disaccharide peak (FIG. 3). This prominentdisaccharide peak in all of the electropherograms was identified asΔUA-GalNAc4S through co-migration of the known dermatan sulfatedisaccharide standards. The two minor peaks that elute around 10 minwere identified as ΔUA-GalNAc2S,4S (*) and ΔUA-GalNAc4S,6S (**),respectively (FIG. 3). A comparison between the ratio of theΔUA-GalNAc4S peak to the total peak area of the mutant digests and therecombinant enzyme showed that H272A and E333A demonstrate fullenzymatic activity over the 12 hr time course of the reaction (Table 2).This suggests that, while His272 and Glu333 are important in the activesite chemistry, chondroitinase B can still function without one of them,albeit at a slower catalytic rate.

TABLE 2 Ratio of ΔDi4S Area to Total Peak Area for Chondroitinase B andMutants Enzyme ΔDi4S: Total Peak Area Chondroitinase B 0.93 K250An.d.^(a) H272A 0.94 E333A 0.93 R363A 0.93 R364A 0.39 ^(a)No peaks wereobserved for the K250A digest.

In contrast, changing Lys250 to alanine completely ablated the activityof chondroitinase B (Table 1 and FIG. 3). To insure that the mutatingLys250 did not influence the overall stability of the protein, the CDspectrum of K250A was compared to the spectrum of recombinantchondroitinase B. While the virtual identity of the CD profiles does notpreclude the possibility that there are perturbations in the localenvironment surrounding Lys250 that are not represented in the CDprofile, it does suggest there are no gross conformational changesinduced in chondroitinase B by mutating Lys250 to alanine (FIG. 5).Therefore, Lys250 plays a role in the catalytic activity ofchondroitinase B.

Along with the active site residues discussed above, we mutated Arg271to alanine. Interestingly, the R271A mutant was expressed at comparablelevels to the recombinant chondroitinase B, but was insoluble. Severalattempts to denature and refold the mutant using different methodsincluding a strong chaotropic agent (4M guanidinium HCl) provedunsuccessful. The insolubility of the R271A mutant could implicate thisresidue in the active site chemistry of chondroitinase B. Anotherpossibility is that removing the side chain of Arg271 somehow interfereswith the hydrophobic stacking interactions of Phe296 and Trp298 leadingto a dramatic decrease in the stability of chondroitinase B (FIG. 1,(B)). In addition to catalytic residues discussed above, two basicresidues proximal to subsites −2 and −1, Arg363 and Arg364, wereselected for mutagenesis based on their potential role in substratebinding. The R363A mutant had a k_(cat) of 404 s⁻¹, leading to a slightincrease in k_(cat)/K_(m) when compared to the recombinantchondroitinase B (Table 1). This twofold increase in k_(cat)/K_(m)suggests that removal of Arg363 allows for a slight increase incatalytic efficiency in chondroitinase B. The R363A mutant produced asimilar profile to chondroitinase B after exhaustive digestion ofdermatan sulfate (FIG. 4).

In contrast to the R363A results, mutating Arg364 to alanine led to acomplete loss of activity in the real-time kinetic assay and an alteredproduct profile after exhaustive digestion of dermatan sulfate (Table 1and FIG. 4). In fact, the ratio of the ΔUA-GalNAc4S peak area to thetotal peak area was only 0.39, significantly lower the ratio for therecombinant chondroitinase B (Table 2). In addition, the ΔUA-GalNAc4Speak was not the only prominent peak in the electropherogram (FIG. 4).

To further characterize the novel peaks seen the R364A digest ofdermatan sulfate, the sample was analyzed using MALDI-MS. Peak 3 had amass of 999.2 Da, which identifies it as a tetrasaccharide containing 3sulfates. Peak 2 had a mass of 1539.7 Da, which identifies it ashexasaccharide containing 5 sulfates. Finally, peak 1 had a mass of1922.4 Da, which classifies it as an octasaccharide also containing 5sulfates. Adding more of the R364A mutant enzyme to the sample did notresult in a significant decrease of these higher order peaks, suggestingthat these oligosaccharides are the end products of the reaction. Assuggested by our structural analysis, Arg364 plays a role in the propersubstrate binding and digestion of dermatan sulfate by chondroitinase B.

Compositional analysis of the DS starting material revealed that theΔUA-GalNAc2S,4S and ΔUA-GalNAc4S,6S disaccharides are 2.3% and 4.6% ofthe total disaccharide content. Interestingly, there is a shift in thepercentages to 5.5% and 2.3% for the ΔUA-GalNAc2S,4S and ΔUA-GalNAc4S,6Sdisaccharides, respectively, when DS was digested by the R364A mutantsuggesting that the oversulfation of the higher order oligosaccharidesis at the 6-O position. Therefore, it appears that Arg364 is involved inchondroitinase B's ability to recognize and cleave regions containingΔUA-GalNAc4S,6S in dermatan sulfate.

Taken together, these results, for the first time, directly implicateLys250, His272, Glu333, and Arg271 in the catalytic degradation ofdermatan sulfate by chondroitinase B. Since the H272A mutation shows a6.5 fold decrease in k_(cat), this residue can be potentially involvedin the proton abstraction (Table 1). Histidine has been implicated inthe enzymatic degradation of other GAG degrading enzymes including GroupB Streptococcal hyaluronate lyase and heparinases I, II, and, III(Pojasek, K., Shriver, Z., Hu, Y., and Sasisekharan, R. (2000)Biochemistry 39, 4012-9; Lin, B., Averett, W. F., and Pritchard, D. G.(1997) Biochem. Biophys. Res. Commun. 231, 379-82; and Shriver, Z., Hu,Y., and Sasisekharan, R. (1998) J. Biol. Chem. 273, 10160-7). However,since the enzyme activity is not completely ablated another residue mayalso be involved in the abstraction of the C5 proton. Glu333, anothercandidate for C5 proton abstraction, showed a nearly 40-fold decrease ink_(cat)/K_(m) when mutated to alanine (Table 1). Nevertheless, since theenzyme still retained close to full activity over a 12 hr period (FIG.3), Glu333 also may not be the sole residue involved in the C5 protonabstraction. One possibility is that Glu333 and His272 work in concertwith one another to both lower the pKa of the C5 proton and to abstractit. Another possibility is that Glu245, the symmetrical active siteresidue to Glu333, may also play a part in the proton abstraction (FIG.1, (B)).

Mutating Lys250 to alanine led to a complete loss of enzymatic activityof chondroitinase B towards the dermatan sulfate substrate. Since theε-NH₂ of the lysine (pKa of 10.5) is mostly protonated in the reactionbuffer (pH 8.0), it seems unlikely that this residue would be involvedin proton abstraction. Also, our conformational study points to theinvolvement of Lys250 in stabilizing the charge of the carboxylatemoiety. Therefore, the loss of enzymatic activity in the K250A mutant ismost likely due to this lack of stabilization of the carboxylate group(and the carbanion intermediate) effectively preventing abstraction ofthe C5 proton.

Materials and Methods

Materials

Dermatan sulfate from porcine intestinal mucosa, glucuronic acid, andgalacturonic acid were purchased from Sigma. Caffeic acid and sodiumtetraborate were purchased from Fluka. Chondroitinase ABC was purchasedfrom Seikagaku/Associates of Cape Cod (Falmouth, Mass.). ChondroitinaseB and the R364A mutant were recombinantly expressed in E. coli andpurified as described previously (Pojasek, K., Raman, R., Kiley, P.,Venkataraman, G., and Sasisekharan, R. (2002) J Biol Chem 277, 31179-86;Pojasek, K., Shriver, Z., Kiley, P., Venkataraman, G., and Sasisekharan,R. (2001) Biochem Biophys Res Commun 286, 343-51). Proteinconcentrations were calculated using the Bradford assay (Bio-Rad) withbovine serum albumin as a standard. All other reagents used are fromcommon sources or are as noted.

Isolation of Defined DS Oligosaccharides

Dermatan sulfate was suspended in 50 mM Tris-HCl, pH 8.0 at aconcentration of 10 mg/ml. To complete the partial digestion of the DS,150 μg of the R364A recombinant chondroitinase B mutant was added to 10ml of the DS solution. The reaction was incubated at 30° C. for 16 hr.The amount of R364A added to the reaction mixture was optimized using CEto ensure a maximal range of partially digested DS reaction products.Upon completion of the reaction, the DS products were separated on a2.5×120 cm Bio-gel P6 column (Bio-Rad) with 500 mM ammonium bicarbonateas the mobile phase. Fractions with an absorbance at 232 nm, the λ_(max)for the Δ^(4,5) double bond formed in the DS product by chondroitinaseB, were pooled corresponding to the peaks containing various length DSoligosaccharides and lyophilized to dryness. The oligosaccharide poolswere re-suspended in water and further fractionated by HPLC using a4.6×250 mm Sphereclone 5 μm amine column (Phenomonex, Torrance, Calif.)with a gradient of 0.1 M to 1.0 M sodium phosphate, pH 4.5 over 30 min.Peaks were collected and desalted on a 2.5×55 cm Bio-gel P2 column(Bio-Rad) with a mobile phase of 500 mM ammonium bicarbonate. Fractionswith absorbance at 232 nm were pooled, lyophilized to dryness, andre-suspended in water.

Semicarbazide Derivitization

The reducing end of the DS oligosaccharides was specifically derivatizedwith semicarbazide to provide a mass tag for MALDI-MS and to produce analtered migration time in the CE. Oligosaccharide solutions were mixed1:1 (v/v) with 50 mM semicarbazide in 60 mM Tris/acetic acid, pH 7.0(Rhomberg, A. J., Shriver, Z., Biemann, K., and Sasisekharan, R. (1998)Proc Natl Acad Sci USA 95, 12232-7). Reactions were heated at 40° C. for16 hr and then analyzed using CE. The percent completion of eachreaction was calculated using the ratio of the peak areas for theproduct and the unlabeled substrate in the CE.

Enzymatic Digests

Enzymatic digests were completed by adding 1 μl of varying dilutions ofchondroitinase B (10-100 nM) or R364A (370 nM) to 15 μl reaction.Reactions were performed in 50 mM Tris-HCl, pH 8.0 with substrateconcentrations ranging from 100-200 μM. The reactions were incubated at30° C. for defined periods of time and heat inactivated at 85° C. for 5min. The reaction products and substrate were analyzed using CE andMALDI-MS as described below without any further sample preparation.

Uronic Acid Plate Assay

A 96 well plate assay was used for determining the relative amount ofuronic acid in a DS oligosaccharide sample (van den Hoogen, B. M., vanWeeren, P. R., Lopes-Cardozo, M., van Golde, L. M., Barneveld, A., andvan de Lest, C. H. (1998) Anal Biochem 257, 107-11). Standards ofgalacturonic acid (GalA) and glucuronic acid (GlcA) ranging from 0-10 μgin a total volume of 40 μl water were added to the standard wells.Varying volumes of each of the DS oligosaccharide samples were dilutedinto 40 μl for comparison to the GalA and GlcA standards. 200 μl ofsodium tetraborate in concentrated sulfuric acid was added to each welland mixed by pipetting. The plate was incubated at 80° C. for 1 hr.After the incubation, the plate was cooled to room temperature and 40 μlof a 1:100 dilution of 100 mg/ml 3-phenylphenol in DMSO with 80%sulfuric acid (v/v) in water was added to each well. The plate wasincubated at room temperature for 15 min. and the color change wasanalyzed in a UV plate reader at λ_(abs) of 540 nm. The absorbance ofthree different amounts of each oligosaccharide was compared to thestandard curves to determine the molar concentration of uronic acid ineach sample. The appropriate conversion factor for each length DSoligosaccharide (i.e. 5 moles GlcA/I mole Deca) was used to calculatethe molar concentration of each oligosaccharide sample.

MALDI-Mass Spectrometry

MALDI-MS experiments were completed conditions similar to thosedeveloped for the analysis of heparin/heparan sulfate oligosaccharides(Rhomberg, A. J., Ernst, S., Sasisekharan, R., and Biemann, K. (1998)Proc Natl Acad Sci USA 95, 4176-81). Briefly, a fresh saturated caffeicacid solution (˜12 mg/ml) in 70% acetonitrile was mixed with a molarexcess of basic peptide (arg-gly)₁₅ prior to the 1:10 dilution of theoligosaccharide. Spots were pre-seeded on a stainless steel MALDI plateas previously described (Rhomberg, A. J., Ernst, S., Sasisekharan, R.,and Biemann, K. (1998) Proc Natl Acad Sci USA 95, 4176-81). A 1 μlaliquot of the sample/matrix solution was added to a pre-seeded spot andallowed to dry. MALDI-MS spectra were acquired in the linear mode on aPerSeptive Biosystems (Framingham, Mass.) Voyager Elite time-of-flightinstrument. Delayed extraction was used to increase resolution aspreviously described (Rhomberg, A. J., Ernst, S., Sasisekharan, R., andBiemann, K. (1998) Proc Natl Acad Sci USA 95, 4176-81). Spectra wereexternally calibrated using the signals for the RG₁₅ and the RG₁₅:Decacomplex.

Capillary Electrophoresis

Capillary electrophoresis was performed using similar conditions tothose developed for the separation of heparin/heparan sulfatedisaccharides (Rhomberg, A. J., Ernst, S., Sasisekharan, R., andBiemann, K. (1998) Proc Natl Acad Sci USA 95, 4176-81). Briefly,uncoated fused silica capillaries (i.d. of 75 μm and 1_(tot) of 80.5 cm)coupled with an extended path detection cell were used on aHewlett-Packard ^(3D)CE unit. Oligosaccharides were detected at 232 nmusing an electrolyte solution of 50 mM Tris/phosphoric acid, pH 2.5.Dextran sulfate was added to the buffer to suppress nonspecificinteractions with fused silica wall of the capillaries. Electrophoreticseparation was performed using reverse polarity at a voltage of −30 kV.Peak identities were confirmed by co-migration with known standards. Adilution series of each oligosaccharide was run on the CE to generate aset of standard curves for determining the molar amount of each speciesin a electropherogram.

Results and Discussion

We generated a range of DS-derived oligosaccharides to use as definedsubstrates for the analysis of the mode of action of chondroitinase B.Coupling these defined substrates with the analytical techniques of CEand MALDI-MS, we were able to examine the time-resolved productformation resulting from the action pattern of chondroitinase B. Wefound that chondroitinase B is a non-random, non-processive, endolyticenzyme that preferentially cleaves longer substrates (decasaccharide) ata higher rate when compared to shorter ones (tetrasaccharide). Inaddition, the R364A mutant, previously shown to have decreased reactionkinetics and an altered product profile, also has an altered mode ofaction when compared to chondroitinase B further emphasizing the rolefor this arginine in substrate processing. This work provides a morecomprehensive understanding of the structure-function relationship forthese biologically important polysaccharides.

Enzymatic Generation and Isolation of Defined DS Oligosaccharides

The first step in characterizing the mechanism of action ofchondroitinase B was the generation and isolation of defined DS-derivedoligosaccharides. Porcine intestinal mucosa DS was partially digestedusing the R364A mutant chondroitinase B that was previously shown tohave decreased reaction kinetics allowing for a greater control over therate of the digestion (Pojasek, K., Raman, R., Kiley, P., Venkataraman,G., and Sasisekharan, R. (2002) J Biol Chem 277, 31179-86). The reactionconditions were optimized to provide maximal yield of DS-derivedoligosaccharides ranging from tetra- to dodecasaccharides. After thecompletion of the enzymatic digestion, the reaction products wereseparated on a Bio-gel P6 column yielding six defined peaks (FIG. 6,(A)). Each fraction was further purified using anion exchange HPLC andthe resulting peaks were desalted to yield pure oligosaccharides.

Each oligosaccharide isolated from the six peaks in the P6 profile wasanalyzed using a tandem approach of CE and MALDI-MS to confirm itsidentity, purity, and composition (Table 3). As a representative of thisanalysis, is a CE electropherogram of the major constituent of Peak 2 inthe P6 profile. The single peak in the CE profile clearly indicated thatthe oligosaccharides had been purified to homogeneity (FIG. 6 (B)). Forthe MALDI-MS analysis, the oligosaccharide resulting from Peak 2 wascomplexed with a basic peptide (RG₁₅) and analyzed in the linear mode.The MALDI-MS profile revealed two defined peaks representing theuncomplexed RG₁₅ (3218.9 Da) and the oligosaccharide:peptide complex(5525.9 Da) (FIG. 6(C)). The difference between the masses of the twopeaks (2297.0 Da) confirms that Peak 2 from the P6 profile is adecasaccharide with 5 sulfates. The mass of the decasaccharidecalculated from the MALDI-MS data agrees exactly with the expected mass(Table 3). Compositional analysis of the decasaccharide peak usingchondroitinase ABC revealed that the 4-O-sulfated disaccharide (Di) wasthe sole product confirming the structure in FIG. 7(A).

TABLE 3 DS-derived Oligosaccharides and their masses Oligo- ChemicalComplex Calculated Expected saccharide Structure Mass (Da) Mass (Da)Mass (Da) Di ΔUA-H_(NAc,4S) n.d. n.d. 503.3 Di-sc ΔUA-H_(NAc,4S)-sc n.d.n.d. 560.4 Tetra ΔUA-H_(NAc,4S)—I—H_(NAc,4S) 4316.7 918.8 918.8 Tetra-scΔUA-H_(NAc,4S)—I—H_(NAc,4S)-sc 4192.5 976.7 975.9 HexaΔUA-H_(NAc,4S)—(I—H_(NAc,4S))₂ 4690.9 1378.5 1378.2 Hexa-sc ΔUA- 4650.81435.2 1435.3 H_(NAc,4S)—(I—H_(NAc,4S))₂-sc OctaΔUA-H_(NAc,4S)—(I—H_(NAc,4S))₃ 5057.2 1837.5 1837.6 Octa-sc ΔUA- 5109.81895.0 1894.7 H_(NAc,4S)—(I—H_(NAc,4S))₃-sc DecaΔUA-H_(NAc,4S)—(I—H_(NAc,4S))₄ 5515.9 2297.0 2297.0 Deca-sc ΔUA- 5568.42354.1 2354.1 H_(NAc,4S)—(I—H_(NAc,4S))₄-sc DoDecaΔUA-H_(NAc,4S)—(I—H_(NAc,4S))₅ 5972.8 2755.8 2756.4

The same combination of CE, MALDI-MS, and compositional analysis wasperformed on all of the isolated oligosaccharides to confirm theiridentity and purity. Peak 1 from the P6 profile was a dodecasaccharidecontaining 6 sulfates with a mass of 2755.8 Da (Table 3). Peak 3 was anoctasaccharide with 4 sulfates at an observed mass of 1837.5 Da. Peak 4yielded a hexasaccharide containing 3 sulfates and a mass of 1378.5 Da.Peak 5 was a tetrasaccharide with 2 sulfates with a mass of 918.8 Da.Importantly, all of the masses for the oligosaccharides obtained byMALDI-MS deviated from the expected mass by ≦1 Da (Table 3). Finally,peak 6 was identified as the 4-sulfated disaccharide using CE and wasnot analyzed by MALDI-MS (Table 3).

The use of MALDI-MS was helpful in assigning the identity of each of theoligosaccharides isolated from the partial digest of DS. A computationalexercise completed previously by our group revealed that from only themass of a GAG oligosaccharide of up to a tetradecasaccharide in length,one could assign the oligosaccharide length and the number of sulfatesthat modify it (Venkataraman, G., Shriver, Z., Raman, R., andSasisekharan, R. (1999) Science 286, 537-42). Combining this MALDI-MSanalysis with the CE-based compositional analysis, we were able tounambiguously assign a structure to each of the DS-derivedoligosaccharides (Table 3). Prior NMR analysis of DS-derivedoligosaccharides identified all of the saturated uronic acids as IdoA(Yang, H. O., Gunay, N. S., Toida, T., Kuberan, B., Yu, G., Kim, Y. S.,and Linhardt, R. J. (2000) Glycobiology 10, 1033-9). Therefore, thelogical assumption was made that the structures of the oligosaccharidesin the current study contain IdoA. It is important to note that whileone previous study used MALDI-MS to identify a single DS-derivedhexasaccharide (Ueoka, C., Nadanaka, S., Seno, N., Khoo, K. H., andSugahara, K. (1999) Glycoconj J 16, 291-305), the current studyrepresents the first broad-range application of MALDI-MS for thecharacterization of a diversity of DS-derived oligosaccharides.

Molar Quantitation of CE Data

To develop a more quantitative technique for representing the amount ofthe different oligosaccharide products in a given CE profile, a set ofstandard curves were generated using the uronic acid plate assay (vanden Hoogen, B. M., van Weeren, P. R., Lopes-Cardozo, M., van Golde, L.M., Barneveld, A., and van de Lest, C. H. (1998) Anal Biochem 257,107-11). Glucuronic acid and galacturonic acid (0-21 nmol) were used togenerate standard curves to which each of the DS-derivedoligosaccharides (Di-Deca) was compared, thereby enabling thedetermination the molar concentration of each of the oligosaccharides.The GlcA and GalA standard curves compared well with one another and theuronic acid assay was repeated at least six times for eacholigosaccharide to insure a standard deviation of less than 10%. Inparallel, a dilution series of each oligosaccharide was run on the CE,and the peak areas were plotted as a function of oligosaccharide sampleconcentration. These experiments yielded a set of standard curves thatenable the direct conversion of a CE peak area into a molarconcentration of that oligosaccharide in a sample. Using these standardcurves, the molar amount of each reaction product, as well as eachsubstrate, was calculated for all of the enzymatic reactions describedbelow.

Mechanism of Action of Chondroitinase B

The 5-sulfated decasaccharide (Deca) was selected as the initialsubstrate for exploring the action pattern of chondroitinase B. Deca'sreasonable length and its two cleavable internal bonds as well as twoexternal bonds make it an ideal substrate for these experiments (FIG. 7,(A)). Enzymatic reactions conditions were optimized such that theproduct profile at a variety of time points could be analyzed using CE.Ultimately, 300 nM chondroitinase B was incubated with 220 μM Deca at30° C. Aliquots were removed at varying time points ranging from 10 s to120 min, heat inactivated, diluted, and analyzed by CE. The peak areasfor the different reaction products were used to calculate a molarconcentration for each oligosaccharide that, in turn, was plotted as afunction of time (FIG. 8). Each of the oligosaccharide peaks wereidentified by co-migration with defined oligosaccharide standards andconfirmed by MALDI-MS.

Over the 120 min time course of the experiment, the major productproduced was Tetra with significant yet diminishing amounts of Hexa alsopresent (FIG. 8, (A)). Examination of the products produced during thefirst 60 s of the reaction revealed that Tetra and Hexa were produced inincreasing, nearly equal molar amounts with negligible amounts of Octaand Di produced during this early phase of the reaction (FIG. 8, (B)).Taken together, these results clearly demonstrate that chondroitinase Bis an endolytic enzyme. In fact, a comparison of the amount of Hexa (theproduct of endolytic cleavage) to Octa (the product of exolyticcleavage) produced during the first minute of the reaction yields a 91%endolytic mode of action for chondroitinase B. Additionally, the lack ofDi products implies that chondroitinase B is a non-processive enzyme. Diwould be an obvious reaction product if chondroitinase B continued todegrade a bound oligosaccharide, a pattern that is seen with bothheparinase I and endogalacturonase I from Asperillus niger with theirrespective substrates (Ernst, S., Rhomberg, A. J., Biemann, K., andSasisekharan, R. (1998) Proc Natl Acad Sci USA 95, 4182-7; Pages, S.,Kester, H. C., Visser, J., and Benen, J. A. (2001) J Biol Chem 276,33652-6). Chondroitinase B likely releases the cleavage products aftereach round of degradation with subsequent rebinding initiating the nextround of catalysis in a similar fashion to heparinase II (Rhomberg, A.J., Shriver, Z., Biemann, K., and Sasisekharan, R. (1998) Proc Natl AcadSci USA 95, 12232-7). Importantly, the direct observation of theendolytic action pattern reported here is in agreement with a previousstudy that relied on changes in sample viscosity and gel electrophoresisas indirect measures of the mechanism of action of chondroitinase B(Jandik, K. A., Gu, K., and Linhardt, R. J. (1994) Glycobiology 4,289-96).

Another interesting observation is that the molar concentrations ofTetra and Hexa in the reaction became divergent once the Deca substratehad been depleted. In addition, a rise in the concentration of Diaccompanied the rise in the concentration of Tetra (FIG. 8, (A)). Theseobservations suggest that chondroitinase B prefers longer substrates,such as Deca to shorter ones, such as Hexa. To confirm this observation,each of the oligosaccharides at a concentration of approximately 150 μMwas digested independently with chondroitinase B and the rate of productappearance was measured using CE and corrected for enzyme concentration.Chondroitinase B shows a clear preference for longer oligosaccharideswith the rate of cleavage for Deca being 18-fold higher than the rate ofcleavage for Tetra. In addition, chondroitinase B cleaves Octa at a7-fold higher rate than it cleaves Hexa. This preference for longersubstrates is comparable to what was observed with both heparinase I andII (Ernst, S., Rhomberg, A. J., Biemann, K., and Sasisekharan, R. (1998)Proc Natl Acad Sci USA 95, 4182-7; Rhomberg, A. J., Shriver, Z.,Biemann, K., and Sasisekharan, R. (1998) Proc Natl Acad Sci USA 95,12232-7), hyaluronan lyase from group B streptococci (Baker, J. R., andPritchard, D. G. (2000) Biochem J 348 Pt 2, 465-71), and the endopectatelyases from Erwinia chrysanthemi (Roy, C., Kester, H., Visser, J.,Shevchik, V., Hugouvieux-Cotte-Pattat, N., Robert-Baudouy, J., andBenen, J. (1999) J Bacteriol 181, 3705-9).

Chondroitinase B Digestion of End-Labeled Oligosaccharides

The DS oligosaccharides were labeled at the reducing end withsemicarbazide thereby introducing a mass tag that could be tracked bymass spectrometry (Rhomberg, A. J., Shriver, Z., Biemann, K., andSasisekharan, R. (1998) Proc Natl Acad Sci USA 95, 12232-7). To ensurethe reducing end had been labeled, the reactions were analyzed by bothCE and MALDI-MS. Interestingly, the end-labeled oligosaccharides had anoticeable increase in their migration time in the CE. Theredistribution of the charge density that results from the semicarbazidelabel stabilizing the ring-opened form of the GalNAc likely producesthis observed migration time shift (FIG. 7, (B) and (C)). Capitalizingon this shift in migration time and the relative simplicity of thereaction products from DS, we were able to use the CE to track theformation of the reaction products that contained the reducing endGalNAc labeled with semicarbazide and compare them to products generatedfrom internal cleavage of the oligosaccharide substrate. The reactionproducts are expressed as the fraction of each respective theoligosaccharide species in the electropherogram [i.e.Hexa-sc/(Hexa-sc+Hexa)]. This enabled us to directly assign relativerates of cleavage for the different bonds in up to a decasaccharide bychondroitinase B. In addition, the integration of each oligosaccharidepeak in an electropherogram resulted in significantly more quantitativedata than that produced using MALDI-MS or other MS-based techniques thatare semi-quantitative at best (Rhomberg, A. J., Ernst, S., Sasisekharan,R., and Biemnann, K. (1998) Proc Natl Acad Sci USA 95, 4176-81).Therefore, this CE-based technique represents a significant improvementon previous techniques used to explore the action pattern of otherpolysaccharide degrading enzymes (Rhomberg, A. J., Shriver, Z., Biemann,K., and Sasisekharan, R. (1998) Proc Natl Acad Sci USA 95, 12232-7).

Digestion of Hexa-sc

A pure hexasaccharide with 3 sulfates was labeled with semicarbazideovernight at 40° C. (FIG. 7, (B) and (D)). The efficiency of thelabeling reaction was 95% as determined by CE. The mass observed mass ofHexa-sc was 1435.2 Da as measured by MALDI-MS indicating an increase of57.0 Da (expected increase of 57.1 Da) by the addition of thesemicarbazide tag (Table 3). Compositional analysis of Hexa-sc yieldedDi and Di-sc products at the expected ration of 2:1.

Experiments were performed to determine suitable digestion conditionsunder which the products as well as the substrate were detectable usingCE and MALDI-MS. Recombinant chondroitinase B was added to aconcentration of 170 nM to the labeled hexasaccharide and incubated at30° C. for 3 min. The sample was heat inactivated at 85° C. for 5 min.and then analyzed by CE. Under these reaction conditions, detectableamounts of Tetra and Tetra-sc as well as Di and Di-sc were observed(FIG. 9, (A) and Table 4). A significant amount of the Hexa-sc substrateremained present indicating that the products observed in the CE wereindicative of the initial rate of enzymatic cleavage (FIG. 9, (A)). Theproducts of cleavage at both Site I and Site II in the Hexa-sc are closeto evenly distributed suggesting that chondroitinase B cleaves each bondwith equal efficiency (Table 4). The slight disparity between the highermolar proportions of the unlabeled products compared to the labeledproducts likely results from the cleavage of the remaining unlabeledsubstrate material still present in the starting sample (Table 4 andFIG. 9, (A)). Importantly, the activity of chondroitinase B did not seemto be altered by the presence of the semicarbazide group on Hexa-sc as,a priori, the enzyme would be expected to cleave both internal bonds ofa hexasaccharide with equal efficiency givens its endolytic nature.

TABLE 4 Cleavage of Hexa-sc with Chondroitinase B Cleavage Reaction[Oligosaccharide] Fraction of Site Product μM Species Site I Tetra-sc24.8 0.46 Di 33.4 0.59 Site II Tetra 29.3 0.54 Di-sc 23.9 0.41

Digestion of Deca-sc

A decasaccharide with 5 sulfates was labeled at the reducing end withsemicarbazide (FIG. 7, (C) and (E)). The labeling reaction was 98%complete as indicated by CE. The mass observed mass of Deca-sc was2354.1 Da as measured by MALDI-MS indicating that an increase of 57.1 Da(expected increase of 57.1 Da) by the addition of the semicarbazide tag(Table 3). Compositional analysis of Deca-sc yielded Di and Di-sc in theexpected ratio of 4:1.

Chondroitinase B at a final concentration of 170 nM was incubated withDeca-sc at 30° C. for 30 s, heat inactivated, and analyzed by CE. Afterthe 30 s digestion, a significant amount of the Deca-sc substrate wasstill present, representing 36% of the total peak area in theelectrophoretogram, indicating that the reaction was in its initialphase (FIG. 10, (A)). In agreement with the endolytic mechanism ofchondroitinase B, no Octa or Di products were formed during the initialcleavage of Deca-sc implying that cleavage occurred only at Site II andIII (FIG. 2, (E)). The lack of Di products also suggests that the enzymeis not processive. Interestingly, the product profile suggests thatchondroitinase B prefers to cleave Deca-sc at Site III, the internalbond closest to the reducing end at a threefold higher rate than SiteII, the internal bond closer to the non-reducing end (FIG. 10, (A) andTable 5). This unequal cleavage is in contrast to the Hexa-sc data whereboth bonds are cleaved with equal efficiency by chondroitinase B andimplies that the enzyme is non-random in addition to endolytic (Table4).

TABLE 5 Cleavage of Deca-sc with chondroitinase B Cleavage Reaction[Oligosaccharide] Fraction of Site Product μM Species Site II Tetra 13.30.38 Hexa-sc 13.0 0.34 Site III Tetra-sc 23.1 0.62 Hexa 24.8 0.66

The R364A chondroitinase B mutant A combination of crystal structureanalysis (Huang, W., Matte, A., Li, Y., Kim, Y. S., Linhardt, R. J., Su,H., and Cygler, M. (1999) J Mol Biol 294, 1257-69) and modeling(Pojasek, K., Raman, R., Kiley, P., Venkataraman, G., and Sasisekharan,R. (2002) J Biol Chem 277, 31179-86) previously implicated Arg364 inchondroitinase B in binding DS. Specifically, the basic side chain ofthis amino acid was positioned to make favorable contacts with the 4-0sulfate of the GalNAc occupying the putative −1 subsite inchondroitinase B (Huang, W., Matte, A., Li, Y., Kim, Y. S., Linhardt, R.J., Su, H., and Cygler, M. (1999) J Mol Biol 294, 1257-69; Pojasek, K.,Raman, R., Kiley, P., Venkataraman, G., and Sasisekharan, R. (2002) JBiol Chem 277, 31179-86). Given that 4-0 sulfation is the hallmarkmodification present in DS, Arg364 was speculated to play a criticalrole in determining the substrate specificity of chondroitinase B. Infact, when this residue was mutated to alanine, the resultingchondroitinase B mutant displayed diminished catalytic efficiency and analtered product profile as analyzed by CE (Pojasek, K., Raman, R.,Kiley, P., Venkataraman, G., and Sasisekharan, R. (2002) J Biol Chem277, 31179-86). Therefore, we sought to further examine the effect ofthe R364A mutation on the action pattern of chondroitinase B bydigesting Hexa-sc and Deca-sc with the mutant enzyme.

Hexa-sc was first digested with the R364A mutant. Since the R364A mutanthas a significantly reduced catalytic efficiency compared tochondroitinase B, 370 nM of enzyme was used and the reaction wasincubated for 20 min. at 30° C. The reaction was heat inactivated asbefore and analyzed using CE. After the 2 hr incubation with R364A, thereaction still contained 25% of the initial Hexa-sc substrate and thedistribution of reaction product was noticeably different from thedistribution produced with the recombinant chondroitinase B (FIG. 9, (B)and Table 6). Instead of degrading each bond with equal efficiency asseen with chondroitinase B (Table 4), the product profile suggests thatthe R364A mutant cleaves at Site I with a four-fold higher rate than atSite II as indicated by the 4:1 molar distribution ratio of the products(Table 6). Therefore, the R364A mutant, in addition to having reducedreaction kinetics, also has an altered action pattern on ahexasaccharide substrate.

TABLE 6 Cleavage of Hexa-sc with R364A Cleavage Reaction[Oligosaccharide] Fraction of Site Product μM Species Site I Tetra-sc60.5 0.78 Di 56.8 0.75 Site II Tetra 17.0 0.22 Di-sc 19.5 0.25

Deca-sc was digested with R364A to examine if the differences in theaction pattern seen with the Hexa-sc substrate were replicated with adecasaccharide. As was the case with the Hexa-sc reaction, 370 nM R364Awas incubated with Deca-sc for 1 min to compensate for the reducedcatalytic efficiency of R364A compared to that chondroitinase B.Similarly to the CE profile produced by chondroitinase B, the R364Aproduct profile shows no significant production of Octa or Di species(FIG. 10, (B)). Therefore, the Arg to Ala mutation does not alter theendolytic mechanism or lack of processivity of chondroitinase B.However, in contrast to the chondroitinase B CE profile, the R364Aproduct profile suggests that the mutant cleaves Site II and III atclose to comparable rates as indicated by the nearly equivalent molarratio of the reaction products (Table 7 and FIG. 10, (B)). Therefore,the Arg364Ala mutation alters the preference of chondroitinase B fromcleaving closer to the reducing end of the oligosaccharide at Site IIIto cleaving both of the internal bonds at a comparable rate.

TABLE 7 Cleavage of Deca-sc with R364A Cleavage Reaction[Oligosaccharide] Fraction of Site Product μM Species Site II Tetra 22.60.47 Hexa-sc 20.9 0.42 Site III Tetra-sc 25.1 0.53 Hexa 28.7 0.58

The results above clearly demonstrate that Arg364 is important in thenormal enzymatic processing of DS by chondroitinase B. Comparing theproduct profile of the degradation of Hexa-sc by chondroitinase B withthe R364A mutant, clearly demonstrates that Arg364 contributes importantcontacts with the DS substrate in the −1 subsite that allow for itsnormal positioning in the active site (Huang, W., Matte, A., Li, Y.,Kim, Y. S., Linhardt, R. J., Su, H., and Cygler, M. (1999) J Mol Biol294, 1257-69; Pojasek, K., Raman, R., Kiley, P., Venkataraman, G., andSasisekharan, R. (2002) J Biol Chem 277, 31179-86). Removal of thesecontacts leads to a three-fold increase in cleavage rate at Site IIcompared to Site I (Table 7) suggesting that there is likely anotherresidue(s) in the +1 or +2 subsite responsible for positioning thesubstrate for cleavage (Pojasek, K., Raman, R., Kiley, P., Venkataraman,G., and Sasisekharan, R. (2002) J Biol Chem 277, 31179-86). In fact, theR364A mutant is unable to cleave Tetra as a substrate further implyingthat a balance of contacts between the −1 and the +1/+2 subsites isrequired for the normal catalytic function of chondroitinase B.Furthermore, the altered product profile with Deca-sc confirms thatArg364 is required for normal substrate binding. In fact, removal ofArg364 leads to shift in the action pattern of chondroitinase B fromnon-random to random. Similarly altering a single amino acid inendopolygalacturonase I and II leads to shift from processive to anon-processive mode of action (Pages, S., Kester, H. C., Visser, J., andBenen, J. A. (2001) J Biol Chem 276, 33652-6). However, in this case theR364A mutant retains the non-processive, endolytic mechanism displayedby chondroitinase B.

We have applied the analytical techniques of CE and MALDI-MS to thequantitative analysis of the enzymatic degradation products from thedepolymerization of defined DS-derived oligosaccharides bychondroitinase B. Chondroitinase B degrades polymeric DS substrates in anon-random, non-processive, endolytic mode of action and kineticallyfavors longer substrates to shorter ones. Labeling the reducing end ofdefined hexa- and decasaccharide with semicarbazide provided aconvenient mass tag and altered the migration time of theoligosaccharides in the CE. Using these labeled oligosaccharides, wewere able to demonstrate that chondroitinase B favors endolytic bondscloser to the reducing end of the substrate. In addition, examination ofthe product profile of the R364A mutant revealed that this residue playsa critical role in the binding of DS substrates for catalysis. Removalof Arg364 leads to a random action pattern without altering theendolytic, non-processive function of chondroitinase B.

Each of the foregoing patents, patent applications and references thatare recited in this application are herein incorporated in theirentirety by reference. Having described the presently preferredembodiments, and in accordance with the present invention, it isbelieved that other modifications, variations and changes will besuggested to those skilled in the art in view of the teachings set forthherein. It is, therefore, to be understood that all such variations,modifications, and changes are believed to fall within the scope of thepresent invention as defined by the appended claims.

1. A modified chondroitinase B having an amino acid sequence of themature peptide of SEQ ID NO: 2 or conservative substitutions thereof,wherein at least one residue at a position selected from the groupconsisting of 116, 184, 213, 219, 245, 250, 271, 272, 296, 298, 318,333, 363 and 364 of SEQ ID NO: 2 has been substituted or deleted.
 2. Themodified chondroitinase B of claim 1, having a modified product profile,wherein the modified product profile of the modified chondroitinase B isat least 10% different than a native product profile of a nativechondroitinase B.
 3. The modified chondroitinase B of claim 2, whereinthe modified chondroitinase B has the amino acid sequence of the maturepeptide of SEQ ID NO: 2, wherein at least one amino acid residue hasbeen substituted and wherein the substituted amino acid is at a positionselected from the group consisting of 272, 333and 364 of SEQ ID NO: 2.4. The modified chondroitinase B of claim 1, having a k_(cat) or K_(M)value for a substrate that is at least 10% different than a nativechondroitinase B k_(cat) or K_(M) value.
 5. The modified chondroitinaseB of claim 4, wherein the modified chondroitinase B has the amino acidsequence of the mature peptide of SEQ ID NO: 2, wherein at least oneamino acid residue has been substituted, and wherein the substitutedamino acid is at a position selected from the group consisting of 272,333, 363 and 364 of SEQ ID NO:
 2. 6. A composition comprising a sterileformulation of the modified chondroitinase B of claim 1 and apharmaceutically acceptable carrier.
 7. A method of analyzing a sampleof polysaccharides, comprising: contacting the sample with the modifiedchondroitinase B of claim 1 and analyzing the sample of polysaccharides.8. A method of identifying the presence of a particular polysaccharidein a sample, comprising: contacting the sample with the modifiedchondroitinase B of claim 1 and identifying the presence of a particularpolysaccharide in the sample.
 9. A method of determining the purity of asample of polysaccharides, comprising: contacting the sample with themodified chondroitinase B of claim 1 and determining the purity of thesample of polysaccharides.
 10. A method for determining the compositionof a sample of polysaccharides, comprising: contacting the sample withthe modified chondroitinase B of claim 1 and determining the compositionof the sample of polysaccharides.
 11. An immobilized modifiedchondroitinase B comprising; a modified chondroitinase B as in claim 1,and a solid support membrane, wherein the modified chondroitinase B isimmobilized on the solid support membrane.
 12. The modifiedchondroitinase B of claim 1, wherein the chondroitinase B is asubstantially purified recombinant form.
 13. The modified chondroitinaseB of claim 2, wherein the modified chondroitinase B has a modifiedproduct profile that is at least 20% different than a native productprofile of a native chondroitinase B.
 14. The modified chondroitinase Bof claim 2, wherein the modified chondroitinase B has a modified productprofile that is at least 50% different than a native product profile ofa native chondroitinase B.
 15. The modified chondroitinase B of claim 4,wherein the modified chondroitinase B k_(cat) or K_(M) value is at least20% different than a native chondroitinase B k_(cat) or K_(M) value. 16.The modified chondroitinase B of claim 4, wherein the modifiedchondroitinase B k_(cat) or K_(M) value is at least 50% different than anative chondroitinase B k_(cat) or K_(M) value.
 17. The modifiedchondroitinase B of claim 15, wherein the substrate is aglycosaminoglycan.